CN114644735A

CN114644735A - Purified glass bead and preparation method and application thereof

Info

Publication number: CN114644735A
Application number: CN202011507212.0A
Authority: CN
Inventors: 郭敏; 吴亮; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2022-06-21

Abstract

The invention discloses a purified glass bead, a preparation method and application thereof. The purified glass beads comprise white glass bead bodies, and the outer surfaces of the purified glass beads are combined with a purification medium or can be combined with the purification medium, so that the purified glass beads are used for purifying the fluorescence-labeled target object. The color of the glass beads of the target object combined with the fluorescent marker can be observed, the combination condition of the target object can be observed visually, the visualization of the purification process is realized, and the process monitoring is facilitated. And a large amount of purification media can be further combined through the comb-shaped polymer, the comb-shaped polymer not only can provide a flexible main chain, but also can provide a large amount of branched chains, the combination and elution of protein are facilitated, and the large increase of the number of the purification media can be realized.

Description

Purified glass bead and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a purified glass bead, and a preparation method and application thereof.

Background

The separation and purification of protein is an important downstream link in the production process of biological medicines, and the effect and efficiency of the separation and purification directly influence the quality and production cost of protein medicines. For protein purification, agarose gel and other materials are commonly used as purification columns or purification microsphere carriers at present. In the prior art, for the separation and purification of antibody substances, production technicians mainly separate and purify antibody molecules in fermentation liquor or reaction liquor through a protein A affinity adsorption column. In the protein A column, the specific binding is carried out between the protein A fixed on the carrier and the specific site at the Fc end of the antibody molecule, thereby realizing the specific and high-efficiency separation of the antibody from the solution. At present, the carrier in the commonly used protein A column mainly adopts materials such as agarose gel and the like.

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 protein is combined and 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, which results in a significant reduction in the number of binding sites for the protein and a reduction in purification efficiency.

In the prior art, purification columns for protein separation and purification mainly use covalent coupling to fix the purification medium. Taking protein a as a purification medium and a protein a column for purifying antibody substances as an example, the protein a column mainly adopts a covalent coupling mode to fix the protein a, and the protein a is covalently coupled to a carrier through a cysteine at a C terminal. Although the covalent coupling mode can ensure that the purification medium is firmly fixed on the carrier, after the purification column is used for many 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.

Magnetic materials are mostly adopted as core materials for the purified microspheres, and the color of the purified microspheres is black or close to black. When the purification medium is purified, the binding condition of the target object cannot be observed visually; because the microspheres are dark in color, even when the fluorescent markers are purified, visual observation is difficult.

Therefore, it is necessary to develop a purification carrier that can directly observe the binding state of a target substance (particularly, a fluorescent label), and can increase the binding rate and reduce the retention ratio and the retention time.

Disclosure of Invention

Based on the above background, the present invention provides a purified glass microbead. The purified glass beads comprise white glass bead bodies, and the outer surfaces of the purified glass beads are combined with a purification medium or can be combined with the purification medium, so that the purified glass beads are used for purifying the fluorescence-labeled target object. The color of the glass beads of the target object combined with the fluorescent marker can be observed, the combination condition of the target object can be observed visually, the visualization of the purification process is realized, and the process monitoring is facilitated. And a large amount of purification media can be further combined through the comb-shaped polymer, the comb-shaped polymer not only can provide a flexible main chain, but also can provide a large amount of branched chains, the combination and elution of protein are facilitated, and the large increase of the number of the purification media can be realized.

1. In a first aspect, the present invention provides a purified glass microbead.

The purified glass beads comprise glass bead bodies, the glass bead bodies are white, functional elements are combined on the outer surfaces of the glass bead bodies, and the functional elements are used as purification media or can be further combined with the purification media.

The glass bead bodies are preferably glass beads wrapped by silicon dioxide.

The glass bead body can be solid or provided with a cavity. When the hollow spaces are provided, the number of the hollow spaces is not limited, and the performance can be controlled by controlling the average density of the whole.

In one preferable mode, the glass bead body has a cavity, and the average density of the glass bead body is less than 1g/cm³(ii) a More preferably, the average density of the glass bead body is 0.2-0.6 g/cm³(ii) a In a more preferred embodiment, the glass bead body has an average density of 0.4 to 0.5g/cm³(ii) a In a more preferred embodiment, the glass bead bulk has an average density of 0.4g/cm³。

In a preferred embodiment, the average particle size of the glass bead is selected from any one of the following values or a range between any two values: 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm.

In a preferred embodiment, the glass beads have an average particle size of 10 to 500 μm.

In a preferred embodiment, the glass beads have an average particle size of 10 to 250 μm.

In a preferred embodiment, the glass beads have an average particle size of 10 to 200 μm.

In a preferred embodiment, the glass bead has an average particle diameter of 10 to 150 μm.

In a preferred embodiment, the glass bead has an average particle diameter of 10 to 100 μm.

In a preferred embodiment, the glass bead has an average particle size of 20 to 100 μm.

In a preferred embodiment, the glass bead has an average particle diameter of 50 to 100 μm.

In a preferred embodiment, the glass bead bulk has an average particle diameter of 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, with a variation of ± 20%, more preferably ± 10%.

2. In a second aspect, the invention discloses purified glass microspheres comprising a comb-like polymer coating.

On the basis of the purified glass bead disclosed by the first aspect, a comb-shaped polymer is fixed on the outer surface of a glass bead body, the comb-shaped polymer is provided with a linear main chain and branched chains distributed along the linear main chain, the linear main chain is fixed on the outer surface of the glass bead body, and the tail ends of the branched chains are connected with the functional elements.

In one preferred mode, the comb polymer is an acrylic polymer having a-C (CO-) -C-unit structure, and the-C (CO-) -C-unit structure is one of repeating unit structures.

In a preferred embodiment, the monomer unit of the acrylic polymer includes one of acrylic acid, acrylate, methacrylic acid, methacrylate, or a combination thereof.

In one preferred mode, the linear main chain of the comb polymer is a polyolefin main chain; in one of more preferred modes, the linear backbone of the comb polymer is a polyolefin backbone provided by an acrylic polymer; in one of the more preferred modes, the comb polymer is represented by-CH (CO-) -CH₂-is a repeating unit.

3. The third aspect of the invention discloses a purified glass bead modified by metal ions.

In the purified glass bead disclosed in the second aspect, the functional element is a metal ion.

More preferably, the metal ion is Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof.

In one of the preferred modes, the comb polymer is represented by-CH (CO-) -CH₂-is a repeating unit, the comb polymer binds nickel ions via a pendant-CO- (carbonyl) and a tricarboxy amino group, the pendant-CO-and the tricarboxy amino group forming an amide bond therebetween; more preferably, the tricarboxyamino group is N, N-bis (carboxymethyl) -L-lysine or nitrilotrisA residue of acetic acid.

4. In a fourth aspect, the invention discloses purified glass microspheres of biotin, a biotin analogue, avidin, an avidin analogue or an affinity complex thereof.

On the basis of the purified glass beads disclosed in the second aspect, the functional element is any one of biotin, biotin analogue, avidin analogue, biotin-avidin complex, biotin analogue-avidin complex, biotin-avidin analogue complex, and biotin analogue-avidin analogue complex.

The functional element here can serve both as a purification medium and as a connecting element for further connection of the purification medium.

5. The fifth aspect of the invention discloses a purified glass bead with a replaceable purifying medium.

On the basis of the purified glass microbeads disclosed in the second aspect, purification media are bound to the ends of the branches of the comb-like polymer, and the purification media are linked to the ends of the branches of the comb-like polymer by a linking member containing an affinity complex.

Preferably, the affinity complex is selected from the group consisting of: any one of a biotin-avidin complex, a biotin analogue-avidin complex, a biotin-avidin analogue complex, and a biotin analogue-avidin analogue complex; the order between the two components of the affinity complex is an optional order.

In a more preferred embodiment, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

6. The sixth aspect of the invention discloses a purified glass bead with replaceable affinity protein as a purification medium.

On the basis of the purified glass microspheres disclosed in the second aspect, the branch ends of the comb-shaped polymer are combined with a purification medium; the purification medium is affinity protein and is connected to the branched ends of the comb-shaped polymer through affinity complex interaction; 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, in which the order between the two components is optional; more preferably, the purification media is attached to the branched ends of the comb polymer in the form of a biotin-avidin protein, wherein biotin-avidin forms an affinity complex.

7. The seventh aspect of the invention discloses a purification medium modified purification glass bead.

The purified glass beads disclosed in the first aspect, a purification medium is bonded to the outer surface of the purified glass beads.

Preferably, on the basis of the purified glass microspheres disclosed in the second or fifth aspect, the purification medium is bound to the ends of the branches of the comb-like polymer.

Preferably, the purification medium comprises metal ions, biotin-type tags, avidin-type tags, polypeptide-type tags, protein-type tags, immunological-type tags, or a combination thereof.

In one preferred embodiment, the metal ion is preferably Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof.

In one preferred embodiment, the biotin-type tag is biotin, a biotin analogue capable of binding avidin-analogue, or a combination thereof.

In one preferred embodiment, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

In a more preferable mode, a comb-shaped polymer is fixed on the outer surface of the glass bead body, and the tail end of a branched chain of the comb-shaped polymer is connected with biotin; the purification medium is avidin, and forms affinity complex binding action with the biotin.

In a preferred embodiment, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof.

In a preferred embodiment, the protein-based tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably one, said affinity protein is selected from the group consisting of: protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof.

In a preferred embodiment, the immunological label is either an antibody-type label or an antigen-type label.

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 an antibody VHH fragment of an anti-protein.

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

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 an antibody Fab fragment.

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

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

In a preferred embodiment, based on the purified glass beads disclosed in the second or fifth aspect, a purification medium is bound to the ends of the branches of the comb polymer, and the purification medium is attached to the ends of the branches of the comb polymer in a manner that: covalent bonds, supramolecular interactions, linking elements, or combinations thereof.

In a preferred embodiment, 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 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.

8. The eighth aspect of the present invention discloses a method for preparing the purified glass beads of the second aspect, comprising the steps of:

the method comprises the following steps: providing white glass microspheres; in a preferred embodiment, the glass beads are hollow glass beads.

Step two: and modifying the surface of the glass bead with a silicon dioxide coating to obtain the glass bead wrapped by silicon dioxide, namely the glass bead body.

One of the preferable modes is that the glass beads are added into a mixed solvent of ethanol and water and stirred, ammonia water is added, and an ethanol solution of tetraethyl orthosilicate is dripped to obtain the glass beads wrapped by silicon dioxide.

Step three: and modifying carbon-carbon double bonds on the surface of the glass bead body to obtain the carbon-carbon double bond modified glass beads.

The acrylic molecule is preferably acrylic acid, an acrylate, methacrylic acid, a methacrylate, or a combination thereof; more preferably, the acrylic monomer molecule is sodium acrylate.

One of the preferred modes is as follows: adding the glass beads wrapped by the silicon dioxide into absolute ethyl alcohol, adding a silane coupling agent of a silane coupling agent functionalized by carbon-carbon double bonds, mixing, and reacting to obtain the glass beads modified by the carbon-carbon double bonds. Some preferred examples of the carbon-carbon double bond-functionalized silane coupling agent are: acryloyl-functional (CH)₂CH-CO-) or allyl-functionalized (CH)₂＝CH-CH₂-) a silane coupling agent.

More preferably, the glass microspheres wrapped by the silica are added into absolute ethyl alcohol, and a silane coupling agent KH570 is added, mixed and heated to react, so that the carbon-carbon double bond modified glass microspheres are obtained.

In another preferred mode, adding the glass beads wrapped by the silicon dioxide into absolute ethyl alcohol, adding an aminated silane coupling agent, mixing, and reacting to obtain amino-modified glass beads; further carrying out covalent reaction with acrylic acid molecules to obtain the glass beads modified by carbon-carbon double bonds; more preferably, the aminated silane coupling agent is silane coupling KH 550.

Step four: and (3) carrying out polymerization reaction on acrylic monomer molecules by taking the carbon-carbon double bond as an initiation center to obtain the comb-shaped polymer modified glass beads.

Step five: and chemically modifying the tail end of the branched chain of the comb polymer, and combining with a functional element to obtain the purified glass bead.

8.1. Preparation method of purified glass beads modified by purification medium

In a preferred embodiment, the method for preparing the purified glass beads comprises the following steps:

the method comprises the following steps: providing white glass microspheres; in a preferred embodiment, the glass beads are hollow glass beads;

step two: wrapping a silicon dioxide coating on the surface of the glass bead to obtain a glass bead body;

step three: modifying carbon-carbon double bonds on the surface of the glass bead body to obtain glass beads modified by the carbon-carbon double bonds;

step four: carrying out polymerization reaction on acrylic monomer molecules by taking the carbon-carbon double bond as an initiation center to obtain comb-shaped polymer modified glass beads;

step five: and chemically modifying the tail end of the branched chain of the comb polymer, and combining with a purification medium to obtain the purified glass bead.

In a preferred mode, the coating of silica on the surface of the glass bead refers to a covalent chemical modification of the silica coating on the surface of the glass bead.

8.2. Preparation method of nickel ion modified purified glass beads

the method comprises the following steps: providing glass beads in a white color; in a preferred embodiment, the glass beads are hollow glass beads;

step two: modifying a silicon dioxide coating on the surface of the glass bead to obtain a glass bead body;

step five: activating the tail end of the branched chain of the comb-shaped polymer, carrying out covalent reaction with tricarboxyamine, and chelating nickel ions to obtain purified glass beads, wherein the purified glass beads are nickel ion modified glass beads.

By "activating" is meant either increasing reactivity, including modifying no reactivity to reactive activity, or modifying low reactivity to high reactivity.

Preferred examples of the tricarboxylylamines include, but are not limited to: n, N-bis (carboxymethyl) -L-lysine, nitrilotriacetic acid, and combinations thereof.

Examples of implementations of the chelated nickel ions are: reacting with nickel sulfate.

8.3. Preparation method of purified glass beads modified by biotin or biotin analogue

step five: activating the tail end of the branched chain of the comb polymer, and combining biotin or biotin analogues after chemical modification to obtain the purified glass beads, wherein the purified glass beads are biotin or biotin analogue modified glass beads.

8.4. Preparation method of avidin or avidin analogue modified purified glass beads

step five: activating the tail end of the branched chain of the comb polymer, and chemically modifying the tail end of the branched chain to combine with avidin or avidin analogues to obtain the purified glass beads, wherein the purified glass beads are the glass beads modified by the avidin or avidin analogues.

Preferred examples of the avidin include, but are not limited to: streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.

Independently optionally, step six, further comprising renewing or replacing the purification medium may be accomplished by eluting the avidin or analog thereof under suitable conditions and then re-binding the same or a different avidin or analog thereof.

8.5. Preparation method of purified glass beads modified by replaceable purification medium

step five: activating the tail end of a branched chain of the comb polymer, and combining biotin or biotin analogues after chemical modification to obtain biotin or biotin analogue modified glass beads;

step six: and reacting the biotin or biotin analogue modified glass beads with avidin or an analogue-purification medium covalent conjugate thereof to form an affinity complex connecting element of biotin or an analogue thereof-avidin or an analogue thereof, and obtaining the purified glass beads, wherein the purified glass beads are the purification medium modified glass beads.

A preferred example of the avidin or analogue thereof-purification medium covalent conjugate is an avidin-avidin covalent conjugate, in which case the purified glass beads are avidin-modified glass beads.

9. The ninth aspect of the present invention provides the use of the purified glass microspheres of any one of the first to seventh aspects for purifying fluorescent markers. The fluorescent marker can be bound by a purification medium directly carried by the purified glass beads or a purification medium carried by the modified glass beads, namely, the fluorescent marker is captured; preferably, the fluorescent marker can be specifically combined with a purification medium directly carried by the purified glass microsphere or a purification medium carried after modification.

10. A tenth aspect of the present invention provides a method for purifying a fluorescent protein marker, wherein the method for purifying the fluorescent protein marker employs any one of the purified glass beads of the first to seventh aspects, the purified glass beads are bound with a purification medium, and the fluorescent marker can be specifically bound with the purification medium; after the functionalized glass beads are combined with the fluorescent marker, the color can be observed by naked eyes.

Optionally, the method further comprises renewing or replacing the purification medium in the purified glass microspheres.

The main advantages and positive effects of the invention include:

1. the white glass bead body is adopted, so that the binding condition of the target object can be visually observed after the target object is bound with the fluorescent mark, the visualization of a purification process is realized, and the process monitoring is facilitated.

2. The outer surface of the purified glass microsphere is combined with a purification medium through a special polymer structure. Polymers with linear main chains are covalently fixed on the outer surface of the glass microsphere body, and the polymers are also provided with a plurality of functionalized branched chains, and specific purification media can be combined at the tail ends of the branched chains. Through the structure, a large amount of purification media hung at the side end of the linear main chain of the polymer are provided on the outer surface of the glass bead body, so that the high retention ratio caused by the traditional net structure is avoided, the limitation of the specific surface area is overcome, and a large amount of target binding sites can be provided. Wherein the kind of the purification medium can be selected according to the kind of the substance to be purified. When the purification medium is attached to the branches of the polymer in a non-covalent strong interaction in the form of an affinity complex, further, the branched backbone between the purification medium and the linear backbone of the polymer also has a binding effect of the affinity complex, so that the purification medium can be easily replaced. When the target substance to be separated and purified is an antibody-like protein, the purification medium is usually an affinity protein.

3. The purified glass beads and the preparation method thereof provided by the invention can provide a large amount of purification media: the method comprises the steps of chemically modifying the outer surface of glass beads, providing a plurality of binding sites on the outer surface of the glass beads, then covalently connecting a polymer to a single binding site on the outer surface of the glass beads, covalently connecting the polymer to the single binding site on the outer surface of the glass beads through one end of a linear main chain, distributing a large number of side branched chains along the linear main chain, wherein 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 connecting a specific purification medium to the nascent binding sites of the polymer branched chains according to specific purification requirements so as to capture specific target molecules (particularly biochemical molecules, including but not limited to antibody protein molecules). Furthermore, the single binding site on the outer surface of the glass beads may be covalently bonded to only one linear polymer backbone or may be covalently bonded to two or more linear backbones, so that the retention ratio is preferably not increased due to the accumulation of chains. 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.

According to the structural design, the surface of the glass microsphere is coated by the polymer carrying a large number of branched chain structures, the limitation of specific surface area is overcome, a large number of purified medium binding sites are provided, and the number of purified media which can be bound on the surface of the glass microsphere is multiplied by multiple times, more than ten times, more than one hundred times and even more than one thousand times, so that a target object is bound at high flux; the glass beads can capture target objects (such as target protein) from a mixing system to the glass beads efficiently, and high-flux combination, namely high-flux separation, is realized.

4. The flexibility of the polymer chain can be utilized, the polymer chain can flexibly swing in a reaction and purification mixed system, the moving space of a purification medium is enlarged, the capture rate and the binding capacity of a target object (such as target protein) are increased, the target object is promoted to be rapidly and fully bound, and high efficiency and high flux are realized.

5. The structural design of the invention enables the purified glass beads to realize the high-efficiency elution of the purified target (such as target protein) during the elution, effectively reduces the retention time and retention proportion of the target, and realizes high efficiency and high yield. The purification medium 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 proportion caused by the traditional net structure are avoided; on the other hand, the branched chain of the polymer further plays a space separation role, so that a purification medium can be fully distributed in a mixed system and is far away from the surface of the glass microsphere and the internal skeleton of the polymer, the efficiency of capturing a target object is improved, the retention time and the retention ratio 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 structure design of the invention can not only utilize the high flexibility of the linear main chain, but also have the advantage of high magnification of the number of the branched chains, and better realize the combination of high speed and high flux, and the separation of high efficiency and high proportion (high yield).

6. The purification medium (such as affinity protein) for purifying the glass beads can be connected to the tail end of the polymer branched chain on the outer surface of the glass beads in a non-covalent strong binding force in an affinity compound mode; when the purification medium needs to be updated and replaced, the purification medium can be conveniently and quickly eluted from the microbeads and recombined with a new purification medium, so that the purification performance of the glass microbeads can be quickly recovered, the glass microbeads can be repeatedly regenerated and used, and the separation and purification cost is reduced.

7. The purified glass beads are convenient to operate and use. The glass bead body can be prepared by adopting glass beads with large size (such as average particle size of dozens of microns to hundreds of microns), the glass beads combined with the target object can be suspended in the upper layer of liquid or the liquid, and can also be deposited in the lower layer of the system, the separation is simple, and the glass beads can be conveniently separated from the mixed system.

8. The purified glass beads provided by the invention have wide application, and the purification medium is selective. According to the specific type of the purification substrate, corresponding purification media can be flexibly loaded in the purification glass beads, and the capture of specific target molecules (particularly protein substances) can be realized. Alternative types of purification media include, but are not limited to, metal ions (e.g., nickel ions), biotin or analogs thereof, avidin or analogs thereof, polypeptide-type tags, protein-type tags (e.g., avidin), immunological-type tags (including, but not limited to, antibody-type tags, antigenic-type tags), and the like. For example, affinity proteins can be selected for targeting, and are generally applied to separation and purification of antibody substances on a large scale.

Drawings

FIG. 1 is a schematic illustration of a purified glass microbead. The purification glass bead takes hollow glass beads wrapped by silicon dioxide as a glass bead body, the outer surface of the glass bead body is connected with a comb-shaped polymer, the tail end of a branched chain of the polymer is connected with a functional element, and the functional element can be used as a purification medium and also can be used as a connecting element for further connecting the purification medium. In the figure, the number of polymer molecules (4) is only for the sake of simplicity and illustration, and does not mean that the number of polymer molecules on the outer surface of the glass 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 schematic illustration of a purified glass microbead. The purified glass bead takes solid glass beads wrapped by silicon dioxide as a glass bead body.

FIG. 3 is a schematic view of the outer surface modification of a purified glass microbead. The purified glass bead takes hollow glass beads wrapped by silicon dioxide as a glass bead body, a comb-shaped polyacrylic acid skeleton is grafted on the outer surface of the glass bead body, and side branch chains chelate nickel ions through tricarboxy amino groups.

FIG. 4 is a schematic diagram of a purified glass bead. The purification glass bead takes hollow glass beads wrapped by silicon dioxide as a glass bead body, the outer surface of the purification glass bead body is connected with a comb-shaped polymer, and the tail end of a branched chain of the polymer is combined with a purification medium by taking an affinity compound as a connecting element. The linking member of the affinity complex is exemplified by a biotin-avidin affinity complex.

FIG. 5 shows the results of gel electrophoresis of purified histidine-tagged mEGFP using nickel ion-modified glass beads. Wherein M corresponds to Marker molecular weight. Wherein Glass beads 20, Glass beads 40 and Glass beads 60 each have an average density of 0.2g/cm³、0.4g/cm³And 0.6g/cm³The Glass powder with the average grain diameter of about 10-100 mu m is adopted by the Glass powder to prepare the purified Glass beads.

Nucleotide and/or amino acid sequence listing

1, the nucleotide sequence of mEGFP, 714 bases in length.

SEQ ID No. 2, amino acid sequence of mEGFP, 238 amino acids in length.

SEQ ID No. 3, nucleotide sequence of protein A, length 873 bases.

SEQ ID No. 4, nucleotide sequence of protein G antibody binding region, 585 bases in length.

SEQ ID No. 5, amino acid sequence of the nano antibody anti-eGFP, length 117 amino acids.

SEQ ID No. 6, nucleotide sequence of tamavidin2, 423 bases in length.

SEQ ID No. 7, the nucleotide sequence of mScarlet, 693 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. Experimental procedures without specific conditions noted in the following examples, preferably according to, with reference to, the specific embodiments described herein, may then be followed by conventional conditions, e.g. "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. Where a definition in a reference conflicts with a definition in the present disclosure, the substance, composition, material, system, formulation, species, method, apparatus, etc. identified in the reference does not conflict with that existing in the cited reference.

Microbeads: micron-sized particles having an average particle size of 1 μm to 1000 μm, also referred to as microparticles. The particles are not limited to spherical shapes, and may be non-spherical shapes, such as ellipsoidal shapes, polyhedral shapes, columnar shapes, irregular shapes, and the like. Preferably, the ratio of the longest particle size to the smallest particle size of the particulate matter does not exceed 5; more preferably, the ratio of the longest particle size to the smallest particle size of the particulate matter does not exceed 2.

The "particle diameter" and "diameter" in the present invention mean an average particle diameter and an average diameter unless otherwise specified or specifically limited. The deviation is preferably. + -. 20%, more preferably. + -. 10%

Glass beads: glass beads. Also referred to herein as glass beads. The glass beads of the present invention may be solid or may contain a cavity. The number of cavities, if any, is not particularly limited.

Purifying the glass beads: the glass beads are combined with functional elements on the outer surface and applied to the purification field. The functional element herein may serve as a purification medium or as a linking element capable of further binding a purification medium.

Hollow glass beads: also known as hollow glass microspheres. In the present invention, it means glass beads having a cavity, which have an average density of less than that of solid glass beads as a whole, and more preferably, have an average density of water or less. The density of the solid glass beads is usually 2.4-4.3 g/cm³Within the range of (1).

Hollow glass beads 20: the average density is about 0.2g/cm³The hollow glass beads of (1).

Hollow glass beads 40: the average density was about 0.4g/cm³The hollow glass beads of (1).

Hollow glass beads 60: the average density was about 0.6g/cm³The hollow glass beads of (1).

The term "divisor" used in the present invention may correspond to any one of the ranges of. + -. 25%,. + -. 20%,. + -. 15% and. + -. 10%, unless otherwise specified.

A glass bead body: preferably silica-coated glass beads.

White glass bead body: the "white" is a broad concept, and includes, but is not limited to, opal, silvery white, moonlight white, plain white, rice white, light white, pale white, ivory white, pearl white, fistular onion white, jade white, fish maw white, grass white, gray white, snow white, titanium white, zinc white, brilliant white, Tibetan white, natural white, apple white, and other white systems, as long as fluorescence can be visually observed after capturing the fluorescent marker (i.e., the visualization of the fluorescent marker is not affected by the background color of the glass microsphere body). In addition, it is also permissible to slightly take other light color systems, such as slightly pink or yellow, as long as the visual observation is not affected. The "visual observation" is preferably observable by the naked eye.

Functional element: if not specifically stated, the bonded functional elements bonded on the outer surfaces of the purified glass beads are meant, and include, but are not limited to, functional elements bonded at the ends of the branches of the polymer when the outer surfaces of the glass beads are wrapped with the poly comb-like polymer. The functional element can be used as a purification medium and also as a connecting element. When used as a connecting element, should be reactive or activated to allow further binding of the purification media.

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.).

Comb-shaped: the comb-like structure of the invention has a linear main axis and a plurality of (at least 3) side branches distributed along the main axis. The "comb" is not limited to a two-dimensional planar comb structure, but may include a three-dimensional comb structure. In the comb-shaped polymer, the linear main shaft and the side branch are distributed corresponding to a linear main chain and a side branch. When the comb-shaped polymer is dissolved in a solution, the linear main chain and the side chain of the comb-shaped polymer can form certain spatial distribution according to a random motion mode and present certain hydrodynamic radius.

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 being not particularly limited, preferably capable of providing a linear main chain and an appropriate amount or 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). After the side carboxyl group of the acrylic polymer is functionalized, the-C (COO-) -C-unit structure forms a covalent bond with an adjacent group, such as an amide bond, an ester bond or the like, usually in the form of-C (CO-) -C-, preferably an amide bond.

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.

The linear main chain, linear main chain have the same meaning in the present invention, and may be used interchangeably.

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 such that the flexible oscillation of the linear main chain can be smoothly exerted, preferably without forming a network structure as much as possible, and without causing accumulation of the branched chains and increase in the retention ratio.

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 purification media or linking elements may be attached to the branching points of the polymer by a branched backbone. The cross point of the branched skeleton and the main chain is also the branch point of the extracted branch. For example, the branched backbone between the purification medium and the linear backbone of the polymer, for example, the affinity protein is used as the purification medium, and the affinity protein at the end of the branched polymer chain can be sequentially coupled to avidin, biotin, and propylenediamine (-NH-CH) residue₂CH₂CH₂-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 the branches. In the case of the linear main chain, the other end of the linear main chain is necessarily linked to a branch point in addition to being fixed to one end of the outer surface of the glass microsphere, and thus, is also broadly included in the scope of the "branch end" of the present invention. Thus, the polymer attached to the outer surface of the glass microspheres of the present invention has at least 1 branch point.

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.

Reactive group: refers to a group that is reactive and capable of chemically reacting with other groups to form a chemical linkage. The linkage of the chemical linkage may be covalent or non-covalent. One of the preferred ways to obtain the reactive groups of the polymer branches is the specific binding site.

Functional groups: the reactive groups may be provided directly or after activation. Can be a reactive group, and can also obtain reactivity after being activated. Covalent reactions may occur and supramolecular interactions, such as affinity complex interactions, may be formed.

Covalent functional group: 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 covalently function 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 a connection which is formed by means of at least one connecting 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 means: directly bonded by covalent bonds. The covalent means includes, but is not limited to, dynamic covalent means, 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.

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 conjugate (covalent conjugate): compounds that are linked directly or indirectly by covalent means are also referred to as covalent linkers or covalent linking complexes.

The term "a-or" an "means a chemical linkage, which may be a chemical bond, a linking element, a covalent linkage or a non-covalent linkage, such as a conjugate or a complex referred to in the present invention, including a" - "symbol. For example, "-" in the biotin-avidin complex is non-covalent; for example, "-" in an avidin-purification medium covalent conjugate is covalent; for example, biotin-avidin proteins are either covalent or noncovalent. Specifically, it is determined by the specific molecular design and the nature of the linkage of the two adjacent components.

Avidin-purification medium covalent conjugates (avidin-purification medium covalent linked complexes): the covalently linked compound has avidin at one end and a purifying medium at the other end, and is directly linked by a covalent bond or indirectly covalently linked by a linking member.

Avidin-avidin covalent conjugates E (avidin-avidin covalent linked complexes E, or avidin-avidin complexes E): an avidin-purification medium covalent conjugate with an affinity protein as a purification medium; the compound formed by covalent connection has one end of avidin and the other end of avidin, and the two are directly connected through covalent bonds or indirectly covalently connected through linking groups. Such covalent attachment means include, but are not limited to, covalent bonds, linking peptides, and the like. Such as: Streptavidin-Protein A covalent conjugate, Streptavidin-Protein A fusion Protein, Streptavidin-enhanced green fluorescent Protein-Protein A fusion Protein (Protein A-eGFP-Streptavidin), Protein A-eGFP-Tamvavidin2, Protein G-eGFP-avidin fusion Protein, Protein G-eGFP-Tamvavidin2 fusion Protein, and the like.

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. It should be noted that, when the affinity complex is referred to in the present invention, the order between the two components is an optional order unless otherwise specified or specifically limited.

Target, also called purification substrate, is the material to be separated from the mixed system. The purification substrate in the present invention is not particularly limited; preferably, the purification substrate is a proteinaceous substance (also referred to herein as a target protein).

Fluorescent marker: a substance bearing a fluorescent label.

Fluorescently labeled target: the target to be purified is fluorescently labeled.

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 will generally behave as a group with the function of binding the purification substrate.

Affinity protein: specifically binds to a target protein and has a high affinity binding force, such as protein A, protein G, protein L, modified protein A, modified protein G, modified protein L, and the like.

Protein A: protein A, a 42kDa surface Protein, was originally found in the cell wall of Staphylococcus aureus. It is encoded by the spa gene, the regulation of which is controlled by the DNA topology, cellular osmolarity and a two-component system known as ArlS-ArlR. Due to their ability to bind immunoglobulins, have been used in research related to the field of biochemistry. Can be specifically combined with the Fc of the antibody, is mainly used for purifying the antibody and can be selected from any commercial products. The terms "Protein A", "SPA", and "Protein A" are used interchangeably herein.

Protein G: protein G, an immunoglobulin binding Protein, is expressed in group C and group G streptococci, similar to Protein a, but with different binding specificities. It is a 65kDa (G148 protein G) and 58kDa (C40 protein G) cell surface protein, which is primarily used for antibody purification by specific binding to antibodies or certain functional proteins, and can be selected from any commercially available product.

Protein L: protein L, is limited to those antibodies that specifically bind kappa (. kappa.) light chains. In humans and mice, most antibody molecules contain a kappa light chain and the remaining lambda light chain. Is mainly used for antibody purification and can be selected from any commercial product.

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 exemplified by Streptavidin (Streptavidin, abbreviated as SA), including its protein subunit, its analogs (exemplified by Tamvavidin2, abbreviated as Tam2), its modified product, its mutant, 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.

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-.

In the present invention, "biotin or a biotin analogue" has the same meaning as "biotin or an analogue thereof" and can be used interchangeably.

In the present invention, "avidin or avidin analog" has the same meaning as "avidin or its analog" and may be used interchangeably.

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 tags.

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.

A polypeptide-type tag: the polypeptide-type tag of the present invention refers to a tag containing a polypeptide tag or a derivative of a polypeptide tag. The polypeptide tag refers to a tag of a polypeptide structure consisting of amino acid units, wherein the amino acid can be a natural amino acid or an unnatural amino acid.

Protein type tag: the protein-type tag of the present invention includes a tag comprising a protein tag or a derivative of a protein tag. The protein tag refers to a tag of a protein structure consisting of amino acid units, wherein the amino acid can be natural amino acid or unnatural amino acid.

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 the antibody type tag also include an anti eGFP nanobody that can specifically bind to eGFP protein.

Antigenic tag: the antigenic tag of the present invention refers to a tag containing an antigenic substance, which is capable of specifically binding to an antibody substance.

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 6 XHis, HHHHHHHHHHHH; such as 8 XHis tags, HHHHHHHHHHHHHHHH), 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 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. A fragment of the antibody is, for example, an Fc fragment.

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, or single domain antibodies, have only one heavy chain variable domain (VHH).

scFV: a single chain antibody variable fragment is a small molecule consisting of a variable region of an antibody heavy chain and a variable region of an antibody light chain connected by a peptide chain, and is the smallest functional structural unit with antibody activity.

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. 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.

PNA: peptide nucleic acids, a class of DNA analogs with polypeptide backbones substituted for sugar phosphate backbones, are novel nucleic acid sequence specific reagents. The third-generation antisense reagent is constructed by computer design on the basis of first-generation and second-generation antisense reagents and finally synthesized artificially, is a brand-new DNA analogue, namely a pentose phosphodiester bond framework in DNA is replaced by a neutral peptide chain amide 2-aminoethylglycine bond, the rest is the same as the DNA, and PNA can recognize and combine with DNA or RNA sequence in a Watson-Crick base pairing mode to form a stable double-helix structure. Because PNA has no negative charge and has no electrostatic repulsion with DNA and RNA, the stability and specificity of combination are greatly improved; unlike the hybridization between DNA or DNA and RNA, the hybridization between PNA and DNA or RNA is hardly affected by the salt concentration of the hybridization system, and the hybridization ability with DNA or RNA molecules is far superior to that of DNA/DNA or DNA/RNA, which is characterized by high hybridization stability, excellent specific sequence recognition ability, no hydrolysis by nuclease and protease, and capability of linking with ligand for cotransfection into cells. These are advantages not possessed by other oligonucleotides.

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

Affinity force: substrate solutions of different concentration gradients were used, substrate concentration when glass beads bound only 50% of the substrate.

IVTT: in vitro transcription and translation, an In vitro transcription and translation system, is 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, and 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.

Eluent (taking the target protein as an 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.

Flow-through liquid: and (3) collecting clear liquid after the mixed system containing the target protein and the microbeads for purification are incubated, wherein the clear liquid contains residual targets which are not captured.

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.

KH 570: 3- (methacrylic acid)Acyloxy) propyltrimethoxysilane, also known as gamma-methacryloxypropyltrimethoxysilane, CAS:2530-85-0, an acryloyl-functional silane coupling agent. Structural formula is

KH 550: 3-aminopropyltriethoxysilane, CAS:919-30-2, an aminated silane coupling agent. Molecular formula is NH₂-(CH₂)₃-Si(OCH₂CH₃)₃。

NTA: nitrilotriacetic acid, also known as nitrilotriacetic acid, nitrilotriacetic acid. Positions in the context of the present invention refer to the corresponding residues.

"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.

The purification glass bead comprises a glass bead body, wherein the glass bead body is white, a functional element is combined on the outer surface of the glass bead body, and the functional element is used as a purification medium or can be further combined with the purification medium.

The glass bead bodies are preferably glass beads wrapped by silicon dioxide.

The purified glass microspheres can be obtained by: providing a white glass bead body, and then modifying functional elements on the outer surface of the glass bead through surface chemical modification. For example, the outer surface is modified with reactive groups such as hydroxyl, carboxyl, aldehyde, amino, etc.; the purification media of the invention may also be bound by covalent or non-covalent bonds; functional elements such as biotin may also be bound to the outer surface.

The glass bead body can be solid or provided with a cavity. When cavities are present, the number of cavities is not limited. Compared with solid glass beads, the hollow glass beads with cavities are easier to stir and realize suspension by stirring, so that the micro beads are more fully contacted with a mixing system.

In some preferred examples, the glass bead body is provided with a cavity, and the average density of the glass bead body is less than 1g/cm³(ii) a More preferably, the average density of the glass bead body is 0.2-0.6 g/cm³(ii) a In a more preferred embodiment, the glass bead has an average density of 0.4 to 0.5g/cm³(ii) a In a more preferred embodiment, the glass bead bulk has an average density of 0.4g/cm³. By controlling the proper density, the suspension of the glass microspheres in the mixed system is facilitated, the glass microspheres are more fully contacted with a target object, and the capture efficiency and the binding rate of the target object are improved.

In the present invention, the glass bead body may have any feasible particle size. In view of the fact that the average density of the whole glass microspheres can be adjusted through the cavity, the glass microsphere body can select larger particle size (referring to outer diameter size), so that the requirement on the preparation process is greatly reduced, and the difficulty of subsequent separation operation is reduced.

In some preferred embodiments, the average particle size of the glass bead bulk is selected from any one of the following values or a range between any two of the following values: 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm.

In some preferred examples, the average particle size of the glass bead bodies is selected from 10 to 500 μm.

In some preferable examples, the average particle size of the glass microsphere body is 10-250 μm.

In some preferred examples, the average particle size of the glass bead bodies is selected from 10 to 200 μm.

In some preferred examples, the average particle size of the glass bead bodies is selected from 10 to 150 μm.

In some preferred examples, the average particle size of the glass bead bodies is selected from 10 to 100 μm.

In some preferred examples, the average particle size of the glass bead bodies is selected from 20 to 100 μm.

In some preferred examples, the average particle size of the glass bead bodies is selected from 50 to 100 μm.

In some preferred embodiments, the glass bead bulk has an average particle size of 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, with a deviation of ± 20%, more preferably ± 10%.

In some preferred modes, the average particle size of the glass microsphere is selected from 1 μm to 1 mm.

In some preferred embodiments, the average particle size of the glass bead body may be, for example, 1 μm, 10 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1000 μm, and the deviation may be within a range of ± 25%, ± 20%, ± 15%, ± 10%.

By controlling the average density of the glass microspheres, the glass microspheres can be stably suspended in the liquid phase, and can be stably suspended in a liquid system without continuous stirring.

The manner in which the functional element is attached to the outer surface of the glass bead body is not particularly limited.

The manner of attaching the functional element to the outer surface of the glass bead body includes, but is not limited to: a covalent bond, a supramolecular interaction, or a combination thereof.

In some preferred examples, the comb polymer is an acrylic polymer having a-C (CO-) -C-unit structure, and the-C (CO-) -C-unit structure is one of repeating unit structures.

In some preferred examples, the monomer unit of the acrylic polymer comprises one of acrylic acid, acrylate, methacrylic acid, methacrylate or a combination thereof.

In some preferred examples, the linear backbone of the comb polymer is a polyolefin backbone; in one of more preferred modes, the linear backbone of the comb polymer is a polyolefin backbone provided by an acrylic polymer; in one of the more preferred modes, the comb polymer is represented by-CH (CO-) -CH₂-is a repeating unit.

A typical purified glass bead based on hollow glass beads is shown in figure 1.

A typical purified glass bead based on solid glass beads is shown in figure 2.

The glass microspheres can be stably suspended in the liquid phase by controlling the particle size, average density, and chemical and structural parameters of the polymer. And can be stably suspended in a liquid system without continuous stirring. On one hand, the particle size of the glass microsphere can be selected in a larger range by adjusting the preparation process of the cavity, on the other hand, the grafting density of the polymer on the outer surface of the glass microsphere can be adjusted, and the characteristics of the polymer such as hydrophilicity, structure type, hydrodynamic radius, chain length, branched chain number, branched chain length and the like can be adjusted, so that the suspension performance of the glass microsphere in the system can be better controlled, and the glass microsphere can be fully contacted with a mixed system containing a target object.

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 sites of the target capture object provided by the invention only utilize the outer surface space of the purified glass beads, 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.

The manner in which the functional element is attached to the end of the branch of the polymer is not particularly limited.

The manner in which the functional element is attached to the end of the branch of the polymer includes, but is not limited to: covalent bonds, 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 modes, 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 the functional element by covalent bonds based on covalent functional groups, which covalently bond the functional element to the ends of the polymer branches. Can be obtained by covalent reaction of the functional covalent groups contained in the branched chains of the polymer molecules on the outer surface of the glass microspheres and the functional elements. Among them, one of the preferred embodiments of the covalent functional groups is a specific binding site.

The covalent bond based on the covalent functional group refers to a covalent bond formed by the covalent functional group participating in covalent coupling. Preferably, the covalent functional groups are carboxyl, hydroxyl, amino, mercapto, a salt of carboxyl, a salt of amino, formate, or any combination of the foregoing covalent 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 covalent functional groups" refers to all branches of all polymer molecules of the outer surface of one glass microbead, allowing participation in the formation of covalent bonds based on different covalent functional groups.

2.1. Polymer structures providing a large number of branch ends

In the purified glass bead provided by the second aspect, the outer surface of the glass bead 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 glass bead body, and the other end of the polymer is free from the outer surface of the glass bead body.

The term "immobilized" refers to "immobilized" on the outer surface of the glass bead body by covalent bonding.

In some preferred embodiments, the polymer is covalently coupled to the outer surface of the glass bead body directly or indirectly through a linking member.

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).

For the glass bead, one end of the polymer is covalently coupled to the outer surface of the glass bead body, the rest ends including all branched chains and all functional elements are dissolved in the solution and distributed in the outer space of the glass bead body, and the molecular chain (particularly the linear main chain) can be fully stretched and swung, so that each branched chain can be fully contacted with other molecules in the solution, and the capture of a target object can be enhanced. When the target object is eluted from the glass beads, the target object can directly get rid of the constraint of the glass beads 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 object, and reduce the retention ratio and the retention time of the target object during elution.

2.2. Linear backbone of comb polymers

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-based monomers, or a polymerization product of a combination thereof (a backbone provided by a copolymerization product thereof), or a backbone of a copolymerization product formed by polymerization of the above-mentioned 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.

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. The polyolefin main chain may be a main chain (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 main chain of the polymer is a condensation polymerization type main chain. 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 type main chain can be a homopolymerization type or a copolymerization 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 glass bead 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 glass bead 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 glass bead body, and a larger movement space is provided for the linear main chains as much as possible.

2.3. Branches of comb polymers

The number of the branched chains is related to factors such as the size of the glass bead 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 glass bead body and the like.

The number of polymer branches is plural, at least 3. The number of side branches is related to factors such as the size of the glass beads, the length of the polymer backbone, the linear density of the side branches along the polymer backbone, and the chain density of the polymer on the outer surface of the glass beads. The amount of polymer branches can be controlled by controlling the feed ratio of the raw materials.

The comb polymer has at least 3 branches.

Each branch end is independently bound or unbound to the purification medium.

When the branch ends are bound to a purification medium, each branch end is independently bound to the purification medium directly or indirectly through a linking member.

When the branched chain ends are bound with the purification media, the number of the purification media may be 1 or more.

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

In one of the preferred modes, the comb polymer is represented by-CH (CO-) -CH₂-is a repeating unit, said comb polymer binding nickel ions via a pendant-CO- (pendant carbonyl) and a tricarboxy amino group, said pendant-CO-forming an amide bond with said tricarboxy amino group; more preferably, the tricarboxyamino group is the residue of N, N-bis (carboxymethyl) -L-lysine or nitrilotriacetic acid.

A typical purified glass bead with nickel ions as the purification medium is shown in fig. 3.

In the purified glass microbeads disclosed in the second aspect, a purification medium is bound to the ends of the branches of the comb-like polymer, and the purification medium is linked to the ends of the branches of the comb-like polymer by a linking member containing an affinity complex.

A typical purified glass bead with replaceable purification media is shown in fig. 4.

Preferably, the affinity complex is selected from the group consisting of: any one of a biotin-avidin complex, a biotin analogue-avidin complex, a biotin-avidin analogue complex, and a biotin analogue-avidin analogue complex; the order between the two components of the affinity complex is an optional order. Taking the biotin-avidin complex as an example of the linking member, either the side of biotin close to the branch point of the polymer (in this case, the side of avidin close to the purification medium) or the side of avidin close to the branch point of the polymer (in this case, the side of biotin close to the purification medium) may be used.

When the purification medium is connected to the terminal of the polymer branch chain of the purified glass microsphere of the invention in a reversible manner such as supramolecular interaction (especially affinity complex interaction), dynamic covalent bond and the like, the purification medium can be eluted from the terminal of the polymer branch chain under appropriate conditions, and then new purification medium is recombined.

And (4) updating the purification medium, wherein the types of the purification medium before and after updating are the same corresponding to the regeneration of the purified glass beads.

The change of the purification medium corresponds to the change of the purified glass beads, and the types of the purification medium before and after the change are different.

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 action of a common non-covalent bond and weaker than the action of a covalent bond, so that the purification medium can be firmly bound at the tail end of a polymer branched chain on the outer surface of the glass microsphere, 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, and then an activation site which can be recombined with a new avidin-purification medium covalent bond (such as a purification medium with a streptavidin label) is released, so that the purification performance of the glass microsphere is quickly recovered, and the separation and purification cost of a target object is greatly reduced. The process of eluting the glass beads modified with the purification medium to remove the avidin-purification medium covalent bonds and thereby recovering the biotin-modified glass beads is referred to as the regeneration of the purified glass beads. The regenerated purified glass beads have released biotin active sites and are capable of re-binding avidin-purification-medium covalent bonds, and the purification-medium-modified glass beads (corresponding to the regeneration of the purified glass beads) are obtained again, so that fresh purification medium can be provided and new target binding sites can be provided. Therefore, the purified glass beads can be recycled, namely the purified medium can be replaced for reuse.

On the basis of the purified glass microspheres disclosed in the second aspect, the branch ends of the comb-shaped polymer are combined with a purification medium; the purification medium is affinity protein and is connected to the branched ends of the comb-shaped polymer through affinity complex interaction; 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, with the order between the two components being an optional order; more preferably, the purification media is attached to the branched ends of the comb polymer in the form of a biotin-avidin protein, wherein biotin-avidin forms an affinity complex.

According to the purified glass bead provided by the aspect, by taking a biotin-avidin connection mode as an example, avidin can be firmly bound to the tail end of a polymer branched chain on the outer surface of the glass bead, and avidin (such as streptavidin) can be eluted from a specific binding position of the biotin to realize synchronous separation of the avidin when the avidin needs to be replaced, so that an activation site capable of being re-bound with a new avidin-avidin covalent binding substance E (such as avidin with a streptavidin label) is released, the purification performance of the glass bead is quickly recovered, and the antibody separation and purification cost is greatly reduced. And (3) eluting the glass beads modified with the avidin, and removing the avidin-avidin covalent conjugates E, thereby recovering the biotin-modified glass beads, which is called the regeneration of the biotin-modified glass beads. The regenerated purified glass beads have released biotin active sites and can be recombined with avidin-avidin covalent conjugates E to obtain avidin-modified glass beads again (corresponding to the regeneration of the purified glass beads), so that fresh avidin can be provided and new antibody binding sites can be provided. Therefore, the purified glass beads of the invention can be recycled, namely, the affinity protein can be replaced and then the purified glass beads can be reused.

7. The seventh aspect of the invention discloses a purification media modified purified glass bead.

On the basis of the purified glass beads provided by the first aspect, a purification medium is bound to the outer surfaces of the purified glass beads.

Preferably, on the basis of the purified glass beads disclosed in the second or fifth aspect, comb-like polymers are fixed on the outer surfaces of the purified glass beads, and purification media are bound to the ends of the branches of the comb-like polymers.

Purification media (purification element)

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. Therefore, substances that can be used for the target protein purification tag can be used as an alternative to the purification medium; peptides or proteins used as purification media may also be used as an alternative to purification tags in the protein of interest.

7.1. Type of purification Medium

The purification medium may contain, but is not limited to, metal ions, biotin-type tags, avidin-type tags, polypeptide-type tags, protein-type tags, immunological-type tags, or combinations thereof.

In some preferred forms, the purification medium is: a metal ion, avidin, an avidin analog that can bind biotin or an analog thereof, biotin, a biotin analog that can bind avidin or an analog thereof, an affinity protein, an antibody, an antigen, DNA, or a combination thereof.

In some preferred embodiments, the metal ion is Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof.

In some preferred embodiments, the biotin-type tag is biotin, a biotin analog that binds avidin, or a combination thereof.

In some preferred embodiments, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

In some preferred modes, a comb-shaped polymer is fixed on the outer surface of the glass bead body, and the tail ends of the branches of the comb-shaped polymer are connected with biotin; the purification medium is avidin, and forms affinity complex binding action with the biotin.

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.

In some preferred forms, the purification medium is: a polypeptide tag, a protein tag, or a combination thereof.

In some preferred forms, the purification medium is an affinity protein.

Examples of such affinity proteins include, but are not limited to, protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and the like.

The definition of antibody, antigen, refers to the term moiety, which is understood to also include, but is not limited to, domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., nanobodies, heavy chains lacking light chains, heavy chain variable regions, complementarity determining regions, etc.), epitopes (epitopes), epitope peptides, variants of any of the foregoing, and the like.

In some preferred forms, the polypeptide tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a tag comprising a WSHPQFEK sequence, a tag comprising a variant sequence of WSHPQFEK, a tag comprising a WRHPQFG sequence, a tag comprising a variant sequence of WRHPQFG, a tag comprising an RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof.

In some preferred embodiments, the protein tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably, the affinity protein is selected from the group consisting of protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof.

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 an antibody against green fluorescent protein or a mutant thereof.

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 embodiments, the antibody type tag is an antibody VHH fragment of an anti-protein.

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

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 an antibody Fab fragment.

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

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

For example, the nanobody shown in SEQ ID No. 5 is used as a purification medium.

7.2. Loading mode of purification medium

The manner in which the purification media is attached to the ends of the branches of the comb polymer is not particularly limited.

The attachment means of the purification media to the ends of the branches of the comb polymer 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 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 purification media is attached to the branched ends of the polymer by a linking element comprising an affinity complex, in which case the purification media is attached to the branched ends of the polymer by affinity complex action.

In some preferred embodiments, the affinity complex selection criteria are: the glass bead 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 polymer branch chain, or can be covalently connected to the outer surface of a glass bead body after chemical modification, such as a binding site of the outer surface, the tail end of a linear polymer main chain and the tail end of a comb-shaped polymer branch chain. Such as a combination of: biotin or its analogs and avidin or its analogs, antigens and antibodies, and the like.

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.

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.

In some preferred embodiments, the purification medium is avidin, which binds to biotin, wherein biotin serves as a linking element; wherein, biotin and avidin form affinity complex binding action.

In some preferred forms, the purification medium is linked to avidin or an avidin analog which binds to said biotin or biotin analog, an affinity complex linkage element being formed between said biotin (or biotin analog) and said avidin (or avidin analog).

In some preferred forms, the purification medium is an affinity protein linked to avidin, which binds to biotin; wherein, the biotin and the avidin form affinity complex binding action, and the affinity complex is used as a connecting element.

In some preferred modes, the tail ends of the branches of the comb-shaped polymer are sequentially connected with biotin, avidin and a purification medium. More preferably, the purification medium is an antibody or antigen. 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 forms, the purification media is attached to the ends of the branches of the comb polymer by the following attachment elements: including, but not limited to, nucleic acids, oligonucleotides, peptide nucleic acids, nucleic acid 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.

7.3. Mechanism of action of the purification Medium

The force of the purification medium for capturing the target molecule in the reaction-purification mixed system can be selected from: including but not limited to covalent bonds, supramolecular interactions, combinations thereof.

In some preferred embodiments, the target is bound to the branched ends of the comb-like polymer of the purified glass microbeads according to the second aspect by the following force: biotin-avidin binding, Streg tag-avidin binding, avidin-avidin binding, histidine tag-metal ion affinity, antibody-antigen binding, or a combination thereof. The Streg tag, which mainly includes but is not limited to peptide tags developed by IBA corporation that can form specific binding with avidin or its analogs, usually contains the WSHPQFEK sequence or its variant sequences.

7.4. Purification of the substrate (preferably one of the protein substances)

The purification substrate of the present invention refers to a purification medium of the present invention for capturing an isolated substance, and is not particularly limited as long as the purification substrate can specifically bind to the purification medium of the present invention.

The purified glass beads of the present invention can be used to separate a target from a mixed system thereof. The object is not limited to one substance, and a combination of plural substances is allowed as long as the purpose of purification is to obtain such a composition, or the form of such a composition can satisfy the purification requirement.

When the purification substrate is a protein substance, the purification substrate is also referred to as a target protein.

7.4.1. Purification tags in proteins of interest

The target protein may not carry a purification tag, and in this case, the target protein itself should be capable of being captured by the purification medium carried by the glass beads. For example, the "target protein, purification medium" refers to a combination of "antibody, antigen", "antigen, antibody", "avidin or its analog, biotin or its analog", and the like.

In some preferred embodiments, the protein of interest carries a purification tag that is capable of specifically binding to the purification medium. One, two or more purification tags per molecule of the target protein; when two or more purification tags are contained, the kinds of the purification tags are one, two or more. In addition, tags are considered to be different types of tags as long as they have different amino acid sequences.

The purification tag in the protein of interest may be selected from the group including, but not limited to: a histidine tag, avidin, an avidin analog, a Streg tag (a tag comprising a WSHPQFEK sequence or variant thereof), a tag comprising a WRHPQFGG sequence or variant thereof, a tag comprising a RKAAVSHW sequence or variant thereof, a FLAG tag or variant thereof, a C tag and variant thereof, a Spot tag and variant thereof, a GST tag and variant thereof, an MBP tag and variant thereof, a SUMO tag and variant thereof, a CBP tag and variant thereof, an HA tag and variant thereof, an Avi tag and variant thereof, an affinity protein, an antibody-based tag, an antigen-based tag, and combinations thereof. It may also be selected from the purification tags disclosed in US6103493B2, US10065996B2, US8735540B2, US20070275416a1, including but not limited to Streg tags and variants thereof.

The purification tag may be fused via the N-terminus or C-terminus.

The histidine tag typically contains at least 5 histidine residues, such as a 5 × His tag, a 6 × His tag, an 8 × His tag, and the like.

The octapeptide WRHPQFGG can specifically bind to core streptavidin (core streptavidin).

A Streg tag capable of forming a specific binding interaction with avidin or an analog thereof, said Streg tag comprising WS HPQFEK or a variant thereof. By way of example, WSHPQFEK- (XaaYaWaaZaa)_n-WSHPQFEK, wherein Xaa, Yaa, Waa, Zaa are each independently any amino acid, Xaa Yaa WaaZaa comprises at least one amino acid and (Xaa Yaa WaaZaa)_nAt least 4 amino acids, wherein n is selected from 1-15 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15); (XaaYaWaaZaa)_nSpecific examples of (G)₈,(G)₁₂,GAGA,(GAGA)₂,(GAGA)₃,(GGGS)₂、(GGGS)₃. Streg tags such as WSHPQFEK, WSHPQFEK- (GGGS)_n-WSHPQFEK、WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK、SA-WSHPQFEK-(GGGS)₂GGSA-WSHPQFEK, WSHPQFEK-GSGGG-WSHPQFEK-GL-WSHPQFEK, GGSA-WNHPQFEK-GGGSGSGGSA-WSHPQFEK-GS, GGGS-WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK, etc.

The sequence of the FLAG tag is DYKDDDDK. Examples of variant sequences of the FLAG tag are DYKDHD-G-DYKDHD-I-DYKDDDDK.

The sequence of the Spot tag is PDRVRAVSHWSS.

The C tag comprises an EPEA sequence.

The GST tag is a glutathione S-transferase tag.

The MBP tag refers to a maltose binding protein tag.

The SUMO tag is a known Small molecule ubiquitin-like modifier (Small ubiquitin-like modifier), and is one of important members of the polypeptide chain superfamily of ubiquitin (ubiquitin). In the primary structure, SUMO has only 18% homology with ubiquitin, however, the tertiary structure and its biological function are very similar.

The sequence of the CBP tag is KRRWKKNFIAVSAANRFKKISSSGAL.

The sequence of the HA tag is YPYDVPDYA.

The Avi tag, a known small tag consisting of 15 amino acid residues, is specifically recognized by biotin ligase BirA.

Antibody-based labels, including but not limited to the complete structure of an antibody (complete antibody), antibodies with/without side chain modifications, antibodies with/without glycosylation modifications, antibodies with/without fatty acid chain modifications, domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., nanobodies, heavy chains lacking light chains, heavy chain variable regions, complementarity determining regions, etc.), and the like.

Antigenic class tags include, but are not limited to, the complete structure of an antigen (complete antigen), domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., epitopes, etc.), and the like.

In some preferred embodiments, the target protein is linked to a purification tag at the N-terminus or C-terminus, or to both termini.

Various purification tags, described in this section, can be candidates for purification media in the purified glass beads of the present invention.

7.4.2. Type of the 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, or can 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. As the glyceraldehyde-3-phosphate dehydrogenase, there may be mentioned, for example, Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase, 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.

7.4.3. Mixed system containing target protein

The purified glass beads of the present invention can be used to separate a target protein from its mixed system. 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 mixing system containing the target protein is not particularly limited as long as the purification medium of the purified glass beads 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.

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 6858422, CN109971775A, CN 6856854, CN 6856856854, CN109971775A, CN 6850720720720720720724, CN 2019198813, CN2019112066163, CN2018112862093(CN 109971775A), CN 20191810681518, CN2020100693833, CN2020101796894, CN109971775A, CN2020102693382 and the in vitro cell-free protein amplification system, DNA template and the method of synthesizing the present invention can be constructed as the template of the in vitro protein amplification system, DNA template of the present invention 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 the purified glass beads on one hand, and can also provide a mixed system for testing the separation effect of the target protein on the other hand.

8. The eighth aspect of the invention discloses a method for preparing the purified glass microspheres of the second aspect.

The purified glass microspheres can be prepared by the following steps: (1) providing a body of glass microspheres (commercially available, preparatorily available) that is white and has reactive groups R on its outer surface₁(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 R₁(ii) covalent attachment; (3) and connecting a functional element at the tail end of the branched chain.

The glass bead bodies are preferably glass beads wrapped by silicon dioxide.

With SiO₂The coated glass beads are taken as an example of a glass bead body, and the preparation process of the purified glass beads can be prepared through the following steps: (1) providing SiO₂Wrapping glass beads (the glass beads can be prepared commercially or can be obtained by purchasing glass powder and further processing), and performing 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(ii) a (3) Functional group F for linking functional element to end of branch₁To (3). In this case, the polymer covalently bonded to the glass bead bulk has a linear main chain, and one end of the linear main chain is covalently fixed to the functional group F₁And a plurality of pendant side chains distributed along the polymer backbone. Preferred ways of said functional elements include, but are not limited to: any one of biotin, biotin analogue, avidin analogue, biotin-avidin complex, biotin analogue-avidin complex, biotin-avidin analogue complex, biotin analogue-avidin analogue complex.

A preferred embodiment of the method for preparing the purified glass microspheres comprises the following steps:

the method comprises the following steps: glass beads are provided. The glass beads are white. The glass beads may or may not have a cavity. In a preferred embodiment, the glass beads are hollow glass beads.

The step can adopt a one-step method or a step method.

The acrylic molecule is preferably acrylic acid, an acrylate, methacrylic acid, a methacrylate, or a combination thereof; more preferably, the acrylic monomer molecule is sodium acrylate. According to the research experiment of the applicant, the experimental result shows that compared with acrylic acid as a polymerization monomer, sodium acrylate is adopted as the polymerization monomer, more purification media can be combined, the purification effect of the prepared purified glass beads is better, and the binding force to a target (taking a target protein as an example, specifically taking histidine-tagged eGFP as an example) can be improved by 5-10 times or even more.

In some preferred embodiments, the carbon-carbon double bond is modified by a one-step method: adding the glass beads wrapped by the silicon dioxide into absolute ethyl alcohol, adding a silane coupling agent functionalized by carbon-carbon double bonds, mixing, and reacting to obtain the glass beads modified by the carbon-carbon double bonds. Some preferred examples of the carbon-carbon double bond-functionalized silane coupling agent are: acryloyl-functional (CH)₂CH-CO-), allyl functionalized (CH)₂＝CH-CH₂-) of a silane coupling agent.

In some preferable examples, carbon-carbon double bonds are modified by a step-by-step method, glass beads wrapped by silicon dioxide are added into absolute ethyl alcohol, and an aminated silane coupling agent is added, mixed and reacted to obtain amino-modified glass beads; further carrying out covalent reaction with acrylic acid molecules to obtain the carbon-carbon double bond modified glass beads; more preferably, the aminated silane coupling agent is silane coupling KH 550.

8.1. Preparation method of purified glass beads modified by purification medium

the method comprises the following steps: providing white glass microspheres; in a preferred mode, the glass beads are hollow glass beads;

step four: carrying out polymerization reaction on acrylic monomer molecules by taking the carbon-carbon double bond as an initiation center to obtain the comb-shaped polymer modified glass beads;

8.2. Preparation method of nickel ion modified purified glass beads

step five: activating the tail end of the branched chain of the comb-shaped polymer, performing covalent reaction with tricarboxyamine, and chelating nickel ions to obtain purified glass beads, wherein the purified glass beads are nickel ion modified glass beads.

Preferred embodiments of the chelating nickel ions are, for example, reaction with nickel sulfate.

8.2.1. Examples of embodiments in which carbon-carbon double bond modification is performed using a one-step process

Taking hollow glass beads as a raw material, one preferred embodiment is as follows.

The first step is as follows: white glass beads were provided.

The second step is that: and modifying the surface of the glass bead with a silicon dioxide coating to obtain a glass bead body. Measuring 5-200 mL of hollow glass beads, adding 50-2000 mL (7/3-5/5, v/v, EtOH/H) of mixed solvent of ethanol and water₂O), mechanically stirring at 100-300 rpm, adding 1-40 mL of ammonia water with the mass fraction of 25% -28%, dropwise adding an ethanol solution of tetraethyl orthosilicate (mixing 3-120 mL of tetraethyl orthosilicate and 3-120 mL of ethanol), and completing dropwise adding within 2-40 hours. After the dropwise addition is completed, separating out the hollow glass beads, and washing the hollow glass beads for 3-5 times by using 10-400 mL of absolute ethyl alcohol. Obtaining the hollow glass beads wrapped by silicon dioxide.

The third step: and modifying carbon-carbon double bonds on the surface of the glass bead body to obtain the carbon-carbon double bond modified glass beads. Adding 20-800 mL of absolute ethyl alcohol and 5-200 mL of silane coupling agent KH570 (one end is amino and the other end is acryloyl) into the cleaned hollow glass beads, mechanically stirring at 100-300 rpm, performing oil bath reaction at 50 ℃ for 2-50 hours, and adjusting to 70 ℃ for oil bath reaction for 0.5-10 hours. After the reaction is finished, separating out the hollow glass beads, washing the hollow glass beads for 3-5 times by 10-400 mL of absolute ethyl alcohol, and washing the hollow glass beads for 3-5 times by 10-400 mL of distilled water. Obtaining the carbon-carbon double bond modified hollow glass bead.

Step four: and (3) carrying out polymerization reaction on sodium acrylate by taking the carbon-carbon double bond as an initiation center to obtain the comb-shaped polymer modified glass beads. Adding 20-800 mL of 0.25-2.5 mol/L sodium acrylate solution into the cleaned hollow glass beads, adding 0.75-7.5 mL of 10% ammonium persulfate solution and 75-750 mu L of tetramethylethylenediamine, reacting for 10-60 minutes at room temperature, separating out the hollow glass beads after the reaction is finished, and cleaning for 3-5 times by using 30-1200 mL of distilled water. Obtaining the sodium polyacrylate modified hollow glass beads. The concentration of the sodium acrylate is, for example, 0.25mol/L, 0.3mol/L, 0.35mol/L, 0.4mol/L, 0.45mol/L, 0.5mol/L, 0.55mol/L, 0.6mol/L, 0.65mol/L, 0.7mol/L, 0.75mol/L, 0.8mol/L, 0.85mol/L, 0.9mol/L, 0.95mol/L, 1.0mol/L, 1.1mol/L, 1.3mol/L, 1.4mol/L, 1.5mol/L, 1.6mol/L, 1.7mol/L, 1.8mol/L, 1.9mol/L, 2.0mol/L, 2.1mol/L, 2.2mol/L, 2.3mol/L, 2.5mol/L, etc. The main chain length of the generated comb-shaped polymer is different by adopting different sodium acrylate concentrations, and within a certain length range, the longer the main chain is, the more side chain can be combined with a purification medium, and the stronger the binding force of the prepared purified glass microsphere to a target object is.

Step five: activating the tail end of the branched chain of the comb-shaped polymer, carrying out covalent reaction with tricarboxyamine, and chelating nickel ions to obtain the purified glass beads (the nickel ion modified glass beads). Adding 25-1000 mL of solution X into the cleaned hollow glass beads, adding 0.0125-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.0125-0.5 mol of N-hydroxysuccinimide (NHS), stirring and uniformly mixing at room temperature, carrying out activation reaction for 10-60 min, and converting the branch chain end of the polymer into a lake imide carboxylate group with high reaction activity after activation. Separating out hollow glass beads, and washing for 3-5 times by 25-1000 mL of distilled water; weighing 0.00625-0.25 mol of N, N-bis (carboxymethyl) -L-lysine (CAS: 113231-05-3), dissolving in 25-1000 mL of solution Y (pH7.2-7.5 PBS buffer solution), adjusting the pH of the solution to 7 by using sodium bicarbonate solid powder, adding into the cleaned hollow glass beads, mechanically stirring for 2-30 hours in a water bath at 20-40 ℃, adding 0.000625-0.025 mol of nickel sulfate solid particles into a reaction system, continuously stirring for 1-5 hours, separating out the hollow glass beads, and cleaning for 5-10 times by using 25-1000 mL of distilled water to obtain the target purified glass beads, namely the nickel ion modified hollow glass beads. Adding a proper amount of distilled water to store the hollow glass beads modified by the nickel ions.

The preferred mode of average density of the hollow glass beads is as described in section 1.

The preferred mode of particle size of the hollow glass beads is as described in section 1.

One specific implementation is as follows: using an average density of 0.2g/cm³And hollow glass beads having an average particle diameter of about 100 μm.

One specific implementation is as follows: using an average density of0.4g/cm³And hollow glass beads having an average particle diameter of about 85 μm.

One specific implementation is as follows: using an average density of 0.6g/cm³And hollow glass beads having an average particle diameter of about 70 μm.

Solid glass beads or glass powder can also be used to replace the hollow glass beads to prepare the purified glass beads.

One specific implementation is as follows: using an average density of about 2.5g/cm³And glass powder with a particle size of about 10-100 μm.

8.2.2. Examples of the embodiment of carbon-carbon double bond modification by stepwise method

The first step is as follows: white glass beads were provided.

The second step: and modifying the surface of the glass bead with a silicon dioxide coating to obtain a glass bead body.

The third step: and modifying carbon-carbon double bonds on the surface of the glass bead body to obtain the carbon-carbon double bond modified glass beads.

Measuring 1-1000 mL of aqueous solution of silica-coated glass beads, cleaning with absolute ethyl alcohol, adding 10-300 mL of ethanol solution (5% -50%, v/v%) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) into the cleaned glass beads, reacting for 2-72 hours, and cleaning the glass beads with absolute ethyl alcohol and distilled water to obtain the amino-modified glass beads.

Removing 1.0X 10^-4About 1mol of acrylic acid is added to a solution X with a pH of 4-6 (solution X: an aqueous solution of MES, CAS: 4432-31-9 and NaCl with a final concentration of 0.01-1 mol/L and 0.1-2 mol/L), and 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 to perform an activation reaction for 3-60 min. And adding the solution into a PBS buffer solution with the pH value of 7.2-7.5 and mixed with 0.5-50 mL of amino-modified glass beads, reacting for 1-48 hours, and washing the glass beads with distilled water to obtain the carbon-carbon double bond-modified glass beads.

Step four: and (3) carrying out polymerization reaction on sodium acrylate by taking the carbon-carbon double bond as an initiation center to obtain the comb-shaped polymer modified glass bead. Taking 0.5-50 mL of glass beads modified by carbon-carbon double bonds, adding 0.5-200 mL of 5-30% (w/v) sodium acrylate solution, 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 glass beads with distilled water to obtain the sodium polyacrylate modified glass beads.

Step five: activating the tail end of the branched chain of the comb-shaped polymer, carrying out covalent reaction with tricarboxyamine, and chelating nickel ions to obtain the purified glass beads (the nickel ion modified glass beads). Transferring 0.5-50 mL of sodium polyacrylate modified glass beads into a solution X, adding 0.001-0.5 mol of EDC & HCl and 0.001-0.5 mol of NHS, reacting for 3-60 min, adding a solution Y dissolved with 0.0001-1 mol of N, N-bis (carboxymethyl) -L-lysine (CAS: 113231-05-3, a tricarboxylamine), reacting for 1-48 h, adding 0.0001-1 mol of nickel sulfate solid particles into the reaction system, reacting for 5 min-24 h, and washing the glass beads with distilled water to obtain nickel ion modified glass beads, namely the purified glass beads, which take nickel ions as a purification medium, have the capacity of specifically binding with a target substance marked by a His label, and can separate and purify the target substance marked by the His label.

8.3. A method for preparing purified glass beads modified by biotin or biotin analogues.

Step two: and modifying the surface of the glass bead with a silicon dioxide coating to obtain a glass bead body.

Step three: and modifying carbon-carbon double bonds on the surface of the glass bead body to obtain the glass beads modified by the carbon-carbon double bonds.

A typical preparation method of the purified glass beads comprises the following steps:

step (1): providing a white glass bead body, chemically modifying the glass bead body, introducing amino groups to the outer surface of the glass bead body, and forming the amino group modified glass bead.

In some preferred modes, the glass bead bodies are chemically modified by using a coupling agent.

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

In some preferred modes, the glass bead body is SiO₂The wrapped glass beads are subjected to chemical modification on the glass bead bodies by using a silane coupling agent; the silane coupling agent is in some preferred forms an amino silane coupling agent.

Step (2): and covalently coupling acrylic acid molecules to the outer surface of the amino-modified glass bead by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form the carbon-carbon double bond-modified glass bead.

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

The functional groups of the acrylic monomer molecules and the polymer branches are defined in the section of nouns and terms.

In other preferred embodiments, the functional group F₁Are specific binding sites.

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 to obtain purified glass beads (a biotin or biotin analogue-modified glass bead) to which biotin or a biotin analogue is bound. In the purified glass microbeads prepared, a large number of sites to which biotin or the like can be bound are provided by acrylic polymers (having a polyacrylic acid skeleton).

8.3. Detailed description of the preferred embodiments

One specific embodiment of preparing the purified glass beads from biotin-modified glass beads 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 glass beads wrapped by the silicon dioxide are used as glass bead bodies; firstly, chemically modifying the glass microspheres wrapped by silicon dioxide by using a coupling agent 3-aminopropyltriethoxysilane (APTES, CAS:919-30-2, an amination coupling agent, also a silane coupling agent, more specifically an amination silane coupling agent KH550), introducing amino groups to the outer surfaces of the glass microspheres, and finishing the SiO reaction₂Activating and modifying to obtain the glass beads modified by amino; then covalently coupling immobilized molecules (acrylic acid molecules, which provide a carbon-carbon double bond and a reactive group carboxyl) to the outer surfaces of the glass beads by utilizing a covalent reaction between the carboxyl and the amino, so that the carbon-carbon double bond is introduced to the outer surfaces of the glass beads to obtain the carbon-carbon double bond modified glass beads; then, polymerization reaction of carbon-carbon double bond is utilized to carry out polymerization of acrylic monomer molecules (such as sodium acrylate and acrylic acid), and the polymerization product is covalently coupled to the outer surface of the glass microsphere while the polymerization reaction is carried out, thus finishing the SiO reaction₂Connecting polymers (covalent connection mode) to obtain the glass beads modified by the acrylic polymers; 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, and the main chain of the polymerization product is a lineA main chain of polyolefin, and a plurality 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 to form a network polymer, a porous structure is formed, which affects the elution efficiency of the target.

In some preferred modes, the amount of the acrylic acid used for preparing the carbon-carbon double bond modified glass beads is 0.002-20 mol/L.

In some preferred modes, the amount of the sodium acrylate used for preparing the acrylic polymer modified glass beads is 0.53-12.76 mol/L.

The outer surface of the glass bead body can also adopt other activation modification modes except amination. For example, the aminated glass beads can further react with acid anhydride or other modification molecules, so as to realize chemical modification of the outer surfaces of the glass beads in a carboxylation or other activation modes.

The immobilized molecules are small molecules which are used for covalently fixing the main chain of the polymer to the outer surface of the glass microsphere. 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 glass bead, the other end of the immobilized small molecule can initiate polymerization reaction including homopolymerization reaction or 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 be one of acrylic acid, acrylate, methacrylic acid, methacrylate ester monomer, or a combination thereof.

As 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 one binding site on the outer surface of the glass microsphere, 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 of introducing polymer molecules of other alternatives to the above-mentioned polymers to the outer surface of the glass microspheres: according to the chemical structure of the polymer substitute and the type of the side-chain active groups thereof, selecting a proper activation modification mode of the outer surface of the glass microsphere, the type of immobilized molecules and the type of monomer molecules, and carrying out proper chemical reaction to introduce a large number of active groups positioned on the side chain into the outer surface of the glass microsphere.

Acrylic polymer molecules (such as a comb molecular chain of sodium polyacrylate) are covalently coupled to the outer surface of the glass microsphere, and then activated sites are provided by functional groups at the tail ends of branched chains, or before biotin or biotin analogue molecules are connected, the tail ends of the branched chains of the polymer molecules can be activated according to reaction requirements, so that the polymer molecules have reaction activity and form activated sites; 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-modified glass beads; 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.

8.3.1. Preparation of acrylic Polymer-modified glass microspheres

Preparing amino-modified glass beads: washing the glass beads wrapped by the silicon dioxide with absolute ethyl alcohol, adding an ethanol solution of 3-aminopropyl triethoxysilane (APTES, coupling agent), reacting, washing, and introducing a large amount of amino groups on the outer surfaces of the glass beads to obtain the amino-modified glass beads.

Preparing the glass beads modified by carbon-carbon double bonds: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl) and N-hydroxysuccinimide (NHS) were added to an aqueous solution of acrylic acid to activate carboxyl groups, and the activated product was added to an aqueous solution of glass beads containing amino group modification. Carboxyl activated on acrylic acid and amino on the outer surface of the glass bead form covalent bond connection (amido bond), and a large number of carbon-carbon double bonds are introduced into the outer surface of the glass bead to obtain the carbon-carbon double bond modified glass bead.

Preparation of acrylic polymer-modified glass beads: adding the aqueous solution of acrylic monomer molecules into the carbon-carbon double bond modified glass beads, and adding an initiator to carry out polymerization reaction of carbon-carbon double bonds. Carbon-carbon double bonds in acrylic monomer molecules and carbon-carbon double bonds on the surfaces of the glass beads are subjected to open bond polymerization, and acrylic polymer molecules are covalently bonded to the outer surfaces of the glass beads, wherein the acrylic polymer contains carboxyl groups; the carboxyl-based group may be present as a carboxyl, formate, or the like. 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.

8.3.2. Preparation of Biotin-modified glass microspheres

Adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl) and N-hydroxysuccinimide (NHS) into a solution of acrylic polymer modified glass beads, activating carboxyl functional groups of side branch chains of polymer molecules on the outer surfaces of the glass beads, adding an aqueous solution of propylene diamine, performing coupling reaction, grafting the propylene diamine at the side branch chain carboxyl position of the acrylic polymer molecules, converting the functional groups of the side branch chains of the polymer into amino from carboxyl, and obtaining the polymer glass beads subjected to amination modification

Adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into an aqueous solution of biotin to activate carboxyl groups in biotin molecules, then adding the solution into an aqueous solution containing amination modified polymer glass beads, and covalently bonding biotin at a position of a nascent group (amino group) of a polymer side branch on the outer surface of the glass bead to obtain glass beads, namely the biotin modified glass beads, with a large number of side branch chains of an acrylic polymer respectively connected with the biotin molecules.

8.3.3. Preferred embodiment(s) of the invention

In some preferred embodiments, the method for preparing the biotin-modified glass beads is as follows:

the method of 8.2.1 or 8.2.2 is adopted to prepare the sodium polyacrylate modified glass beads.

Transferring 0.5-50 mL of sodium polyacrylate modified glass beads 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 mixing with polymer of glass microsphere modified by sodium polyacrylateCOONa of the side branch chain is converted into amino; weighing 1.0 × 10^-6～3.0×10^-4Adding biotin into the solution X in mol, adding 2.0X 10^-6～1.5×10^-3mol 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 glass bead solution, reacting for 1-48 hours, and cleaning with distilled water to obtain the biotin-modified glass beads.

step five: activating the tail end of the branched chain of the comb-shaped polymer, and chemically modifying the tail end of the branched chain to combine with avidin or an avidin analogue to obtain the purified glass bead, wherein the purified glass bead is the glass bead modified by the avidin or the avidin analogue.

Preferred examples of the avidin include, but are not limited to, streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.

Reference may be made to 8.3. method for preparing biotin-modified glass beads, using avidin or an analog thereof instead of biotin or an analog thereof as a starting material.

Optionally, step seven, further comprising renewing or replacing the purification medium by eluting the covalent bond of avidin or an avidin analog to the purification medium under suitable conditions and re-bonding the same or a different covalent bond of avidin or an analog thereof to the purification medium.

In some preferred embodiments, the protein A-mEGFP-avidin fusion protein is bound to biotin-modified glass beads and forms a biotin-avidin affinity complex linking element. Examples of the ProteinA-mEGFP-avidin fusion protein include ProteinA-mEGFP-Streptavidin fusion protein and ProteinA-mEGFP-Tamavidin2 fusion protein.

In some preferred embodiments, the protein G-mEGFP-avidin fusion protein is bound to biotin-modified glass beads and forms a biotin-avidin affinity complex linking element. Examples of the ProteinG-mEGFP-avidin fusion protein include ProteinG-mEGFP-Streptavidin fusion protein and ProteinG-mEGFP-Tamavidin2 fusion protein.

In some preferred embodiments, the anti EGFP-avidin fusion protein is bound to biotin-modified glass beads and forms a biotin-avidin affinity complex linking element. In some more preferred embodiments, the anti EGFP-avidin fusion protein is an anti EGFP-mScarlet-avidin fusion protein. In some more preferred modes, the anti EGFP-avidin fusion protein is an anti EGFP-mScelet-Tamvavidin 2 fusion protein, a fusion protein of nanobodies.

The sequence of protein A is derived from Staphylococcus Aureus, SPA for short. The amino acid sequence of SPA is 516 amino acid residues in total length, and amino acids 37-327 are selected as gene sequences used for constructing fusion protein, namely antibody binding domain of SPA. The nucleotide sequence is obtained after the sequence is optimized by an optimization program, and the nucleotide sequence is shown in SEQ ID No. 3.

The nucleotide sequence of ProteinG (SEQ ID No.:4) was from the group G Streptococcus (Streptococcus sp. group G), and the gene sequence of the antibody-binding region thereof was selected.

The mEGFP has a nucleotide sequence shown in SEQ ID NO. 1 and an amino acid sequence shown in SEQ ID NO. 2.

Streptavidin, Streptavidin.

The anti-EGFP, namely the anti-eGFP, is a nano antibody with an amino acid sequence shown as SEQ ID No. 5 and resisting green fluorescent protein.

Tamavidin2, an avidin analogue, is a protein with biotin-binding capacity, contains 141 amino acid units in total, and obtains a nucleotide sequence thereof through codon conversion and program optimization, as shown in SEQ ID No. 6.

The mScalet is a bright red fluorescent protein with the corresponding nucleotide sequence of SEQ ID No.: 7.

8.5.1. Preferably, the replaceable affinity protein is used as a purification medium

In one preferred mode, the biotin-modified glass microsphere material prepared in 8.3 can be combined with avidin-avidin covalent bond E.

Avidin-avidin covalent conjugate E (avidin-avidin covalent conjugate E, also referred to as avidin-avidin conjugate E) is a conjugate formed by covalent conjugation, in which one end is avidin or an analog thereof, and the other end is avidin, and the two are directly linked by a covalent bond or indirectly linked by a covalent linking member. The covalent linking group includes a covalent bond, a linking peptide, and the like. Avidin-binding substance E is exemplified by streptavidin-bearing avidin, wherein the avidin is selected from the group consisting of, but not limited to, protein A, protein G, and/or protein L, and the like. Examples of avidin-avidin binders E also include: Streptavidin-Protein a conjugate, Streptavidin-Protein a fusion Protein, Streptavidin-enhanced green fluorescent Protein-Protein a fusion Protein (Protein a-eGFP-Streptavidin), Protein a-eGFP-Tamavidin2, Protein a-eGFP-Tamavidin1, and the like; wherein the eGFP broadly comprises an eGFP mutant, Streptavidin is Streptavidin, and Tamavidin1 and Tamavidin2 are both avidin analogues.

Avidin binds specifically to biotin to form an affinity complex. The binding effect of the affinity complex between avidin and biotin may be replaced with the binding effect of another affinity complex, and the effect of reusing the affinity protein may be similarly achieved. But more preferably the affinity complex effects provided by avidin and biotin; this is because the two proteins have high specificity and high affinity, biotin has an extra carboxyl group for bonding in addition to the binding domain of avidin, and avidin is also easily prepared as a fusion protein with avidin.

Affinity complex selection criteria: has good specificity and strong affinity, and also provides a site for chemical bonding, so that the covalent bonding can be covalently connected to the tail end of the polymer branch chain or can be covalently connected to the tail end of the polymer branch chain after chemical modification.

8.5.2. Preparation process

In some embodiments, the following step six is performed on the basis of the biotin-modified glass beads prepared in section 8.3 above, to obtain purified glass beads with affinity proteins as purification media.

Step six: a covalent conjugate that binds avidin or an avidin analog to an affinity protein (e.g., avidin-affinity protein covalent conjugate E). And (3) binding the covalent conjugate (such as avidin-avidin covalent conjugate E) to the tail end of the branched chain of the polymer through the specific binding action between biotin or an analogue thereof and avidin or an analogue thereof, and forming an affinity complex between the biotin or the analogue thereof and the avidin or the analogue thereof to obtain the avidin-modified glass bead.

For example: an avidin-avidin covalent conjugate E (for example, streptavidin-bearing avidin, wherein the avidin is selected from the group consisting of but not limited to protein A, protein G and/or protein L, and the like) is added into a system of biotin-modified glass beads, the avidin is non-covalently linked to the polymer branch ends on the outer surfaces of the glass beads by utilizing the extremely strong specific affinity between biotin and avidin (such as streptavidin), thereby obtaining avidin-modified glass beads which can be used for separating and purifying antibody substances, and the avidin is used as a purification medium to provide binding sites for capturing target proteins.

Optionally also independently comprises a seventh step of renewing the avidin-avidin covalent conjugates E, which may be achieved by eluting the avidin-avidin covalent conjugates E under suitable conditions and then re-binding the avidin-avidin covalent conjugates E.

Or independently optionally comprises a seventh step of replacing the avidin-avidin covalent conjugates E, which may be effected by eluting the avidin-avidin covalent conjugates E under suitable conditions, followed by binding a different type of avidin-avidin covalent conjugates E.

8.5.3. Preparation of avidin-protein A-conjugated glass beads (a protein A-modified glass bead)

Biotin-modified glass beads are added to a fusion protein solution of avidin-protein a covalent conjugate E (e.g., protein a-eGFP-Streptavidin, protein a-eGFP-Tamvavidin2), and mixed for incubation. Protein A is fixed on the terminal group of the polymer branch on the outer surface of the glass microsphere through the specific binding of avidin (such as Streptavidin or Tamvavidin2) and biotin, so as to obtain the glass microsphere combined with avidin-protein A. In the obtained protein A modified glass bead structure, a side chain of an acrylic polymer contains an affinity complex structure of biotin-avidin-protein A, the side chain is covalently connected to a linear main chain branch point of the polymer through a biotin end, a non-covalent strong specific binding effect of the affinity complex is formed between the biotin and the avidin, the avidin and the protein A are covalently connected, a fluorescent label can be inserted between the avidin and the protein A, and other connecting peptides can also be inserted.

Among them, avidin-protein A fusion proteins, such as ProteinA-eGFP-Streptavidin fusion protein and ProteinA-eGFP-Tamvavidin2 fusion protein, can be obtained by in vitro cell-free protein synthesis through IVTT reaction. At the moment, the biotin-modified glass beads are mixed with the supernatant obtained after the IVTT reaction, and the binding of the affinity protein A is realized through the specific binding action between the biotin on the outer surface of the glass beads and the avidin fusion protein in the solution.

8.5.4. The binding capacity of the affinity protein on the outer surface of the glass bead can be determined by the following method (taking the fluorescent protein eGFP as an example):

first, after the binding reaction between the solution of the affinity protein and the biotin-modified glass beads was completed, the solution was centrifuged, and the liquid phase was collected and recorded as flow-through liquid. At this time, the concentration of the affinity protein in the liquid phase decreases. The fluorescence intensity of the fluorescent protein eGFP bound on the glass beads is calculated by measuring the change value of the fluorescent protein eGFP in the supernatant obtained by IVTT reaction before and after binding the glass beads, and the concentration of the affinity protein is obtained by conversion. When the concentration of the affinity protein in the flow-through liquid is basically unchanged compared with the concentration of the affinity protein in the IVTT solution before incubation of the glass beads, the adsorption of the glass beads on the affinity protein is saturated, and the fluorescence value of the corresponding fluorescent protein eGFP is not obviously changed. The pure product of eGFP can be used to establish a standard curve of fluorescence value and eGFP concentration, so as to quantitatively calculate the content and concentration of avidin-avidin (such as streptavidin-protein A) bound on the glass beads.

The separation and purification of the antibody by using the protein A modified glass beads can be carried out by calculating the binding capacity of the antibody (taking bovine serum antibody as an example) by the following method: incubating the protein A modified glass beads with a bovine serum antibody solution (obtained by expressing an antibody by using an in vitro protein synthesis system or obtained by the market), eluting the bovine serum antibody from the glass beads by using an elution buffer solution after the reaction is finished, and allowing the separated bovine serum antibody to exist in the eluent. The concentration of Bovine Serum Albumin (BSA) in the eluate was determined by the Bradford method. Meanwhile, BSA is used as a standard protein to carry out enzyme-labeling instrument test, the standard protein is used as a reference, the protein concentration of the purified antibody can be calculated, and the yield of separation and purification are further calculated.

8.5.5. Regeneration of affinity protein-modified glass beads: replacement of purification Medium (example of affinity protein not protein A)

Elution Replacing protein A: the synchronous separation of protein A is realized while the avidin is eluted, so that the avidin-protein A is replaced.

For example: adding a denaturation buffer solution (containing urea and sodium dodecyl sulfate) into the glass beads modified by the protein A, incubating in a metal bath at 95 ℃, washing off avidin-protein A fusion proteins (such as SPA-eGFP-Tamvavidin2) combined with biotin in the glass beads to obtain regenerated biotin-modified glass beads (releasing biotin at the tail end of a polymer branch chain and becoming sites for combining with a purification medium again), adding a fresh solution of the avidin-protein A fusion proteins (such as supernatant obtained after IVTT reaction of SPA-eGFP-Tamvavidin2) into the regenerated biotin-modified glass beads, enabling the released biotin-binding sites of the glass beads to be combined with new avidin-protein A (such as SPA-eGFP-Tamvavidin2), and forming noncovalent specific binding action between the biotin and avidin (such as Tamvavidin2) again, thereby realizing the replacement of the protein A and obtaining the regenerated affinity protein modified glass beads.

9. The ninth aspect of the present invention provides the use of the purified glass microspheres of any one of the first to seventh aspects for purifying fluorescent markers. The fluorescent marker can be bound by a purification medium directly carried by the purified glass microsphere or a purification medium carried by the purified glass microsphere after modification, namely, the fluorescent marker is captured; preferably, the fluorescent marker can be specifically combined with the purification medium directly carried by the purification glass microsphere or the purification medium carried by the purification glass microsphere after modification.

Optionally, the method further comprises renewing or replacing the purification medium in the purified glass beads.

The protein factor system based on the in vitro cell-free protein synthesis method (D2P technique) is used in the following examples. The in vitro protein synthesis system (IVTT system) used in the in vitro cell-free protein synthesis method of the following examples 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 particularly 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 DNA template (the coded protein contains a fluorescent label) with the final concentration of 15 ng/. mu.L 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 20-30 ℃ for reaction for 3-20 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.

The DNA template adopts a nucleic acid template for coding 8 His-mEGFP. A DNA fragment containing an encoding gene of 8His-mEGFP (histidine-tagged mEGFP, wherein the mEGFP is an A206K mutant of eGFP, the nucleotide sequence is shown as SEQ ID No.:1, and the amino acid sequence is shown as SEQ ID No.: 2) is inserted into a plasmid vector by adopting a PCR amplification and homologous fragment recombination method to construct a plasmid vector for expressing the mEGFP. The plasmid was confirmed to be correct by gene sequencing. The components of the plasmid include functional elements such as a T7 promoter, a 5 'UTR, a leader peptide coding sequence, 8 × His (histidine tag), an mgfp coding sequence, a 3' UTR, a LAC4 terminator, f1 ori (replication initiation site), an AmpR promoter, an AmpR gene (ampicillin resistance gene), ori (high copy number replication initiation site), a lacI promoter, and a lacI (LAC repressor) coding gene. And carrying out in-vitro DNA amplification by using the plasmid DNA for coding the 8His-mEGFP as a nucleic acid template by using a phi29 DNA polymerase by an RCA amplification method to obtain the DNA template for coding the 8 His-mEGFP.

Example 1 preparation of Nickel ion-modified hollow glass microspheres 20

The first step is as follows: white glass beads were provided. Providing hollow glass beads 20 having an average density of about 0.2g/cm³The average particle size was about 100 μm, and the appearance was pale white.

The second step is that: and modifying the surface of the glass bead with a silicon dioxide coating to obtain a glass bead body. 50mL of the hollow glass beads 20 were measured, and 500mL of a mixed solvent of ethanol and water (7/3, v/v, EtOH/H) was added₂O), mechanically stirring at 300rpm, adding 10mL of ammonia water with the mass fraction of 28%, dropwise adding an ethanol solution of tetraethyl orthosilicate (mixing 30mL of tetraethyl orthosilicate with 30mL of ethanol), and completing dropwise addition within 20 hours. After completion of the dropwise addition, the hollow glass beads 20 were separated and washed 3 times with 100mL of anhydrous ethanol. Hollow glass beads coated with silica are obtained.

The third step: and modifying carbon-carbon double bonds on the surface of the glass bead body to obtain the carbon-carbon double bond modified glass beads. 200mL of absolute ethanol and 50mL of KH570 as a silane coupling agent were added to the washed hollow glass beads 20, and the mixture was mechanically stirred at 300rpm, reacted in an oil bath at 50 ℃ for 48 hours, and then adjusted to 70 ℃ for 2 hours. After completion of the reaction, the hollow glass beads 20 were separated, washed 3 times with 100mL of absolute ethanol and 3 times with 100mL of distilled water. Obtaining the carbon-carbon double bond modified hollow glass bead.

Step four: and (3) carrying out polymerization reaction on sodium acrylate by taking the carbon-carbon double bond as an initiation center to obtain the comb-shaped polymer modified glass bead. 200mL of a 2.5mol/L sodium acrylate solution was added to the washed hollow glass beads 20, 7.5mL of a 10% ammonium persulfate solution and 750. mu.L of tetramethylethylenediamine were added, and the reaction was carried out at room temperature for 30 minutes, after completion of the reaction, the hollow glass beads 20 were separated and washed 5 times with 300mL of distilled water. Obtaining the comb-shaped polyacrylic acid polymer (sodium salt form) modified hollow glass microspheres.

Step five: activating the tail end of the branched chain of the comb-shaped polymer, carrying out covalent reaction with tricarboxyamine, and chelating nickel ions to obtain the purified glass beads (the nickel ion modified glass beads). Adding 250mL of the solution X into the washed hollow glass beads 20, adding 0.125mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.125mol of N-hydroxysuccinimide (NHS), stirring and uniformly mixing for 30min at room temperature, separating out the hollow glass beads 20, and washing for 3 times by using 250mL of distilled water; weighing 0.0625mol of N, N-bis (carboxymethyl) -L-lysine (CAS: 113231-05-3) and dissolving in 250mL of solution Y, adjusting the pH of the solution to 7 by using sodium bicarbonate solid powder, adding the solution into the washed hollow glass beads 20, mechanically stirring the solution in a water bath at 30 ℃ for 20 hours, adding 0.00625mol of nickel sulfate solid particles into a reaction system, continuing stirring the solution for 2 hours, separating out the hollow glass beads 20, washing the hollow glass beads with 250mL of distilled water for 8 times to obtain the nickel ion modified hollow glass beads 20, and adding a proper amount of distilled water to store the nickel ion modified hollow glass beads 20.

Wherein the content of the first and second substances,

solution X: the final concentrations are 0.01-1 mol/L2-morpholine ethanesulfonic acid (CAS: 4432-31-9) and 0.1-2 mol/L NaCl respectively.

Solution Y: PBS buffer solution pH 7.2-7.5, such as: the final concentrations are 0.0684mol/L disodium hydrogen phosphate, 0.0316mol/L sodium dihydrogen phosphate and 0.15mol/L sodium chloride aqueous solution respectively.

Example 2 preparation of Nickel ion-modified hollow glass microspheres 40

Using the preparation method of example 1, hollow glass beads 40 (average density about 0.4 g/cm) were used³An average particle diameter of about 85 μm and a pale white appearance) was used as a raw material in place of the hollow glass beads 20.

Example 3 preparation of Nickel ion-modified hollow glass microspheres 60

Using the preparation method of example 1, hollow glass beads 60 (average density about 0.6 g/cm) were used³An average particle diameter of about 70 μm and a pale white appearance) was used as a raw material in place of the hollow glass beads 20.

Example 4 preparation of Nickel ion modified solid glass micropowder

The preparation method of example 1 was adopted, and glass frit (density about 2.5 g/cm) was used³An average particle diameter of about 10 to 100 μm, white in appearance and slightly pink) as a raw material in place of the hollow glass beads 20.

EXAMPLE 5 testing of cohesion

Corresponding amounts of the purified glass bead suspensions were taken in centrifuge tubes according to table 1. The liquid was aspirated off and the four glass beads were washed three times with binding buffer (50mM Tris-HCl pH8.0, 500mM NaCl, 5mM imidazole).

Table 1 binding force test parameters for four purified glass microspheres of examples 1-4.

Purified glass beads	Volume of suspension	Volume fraction of glass beads	Volume of the column bed
				Glass powder suspension	20μL	8mL/16.7mL＝47.9％	9.58μL
Hollow glass bead 20 suspension	100μL	7.3mL/83.9mL＝8.7％	8.7μL
				Hollow glass bead 40 suspension	100μL	5mL/106.5mL＝4.7％	4.7μL
Hollow glass bead 60 suspension	100μL	4.5mL/94.5mL＝4.8％	4.8μL

0.2mL of IVTT reaction solution expressing mEGFP (the N end of mEGFP is connected with histidine tag 8His) is taken, centrifuged for 10 minutes at 4000 revolutions and 4 ℃, and the supernatant is taken to measure the RFU value. Then adding the mixture into different centrifuge tubes containing the purified glass beads, fully and uniformly mixing, and performing rotary incubation for 3 hours at 4 ℃.

And (4) performing centrifugal separation, wherein the collected supernatant is the flow-through liquid. The purified glass beads were washed twice with 1mL of a washing solution (50mM Tris hydrochloric acid, pH8.0, 500mM sodium chloride, 20mM imidazole), and finally eluted with 80. mu.L of an eluent (50mM Tris hydrochloric acid, pH8.0, 500mM sodium chloride, 250mM imidazole). The washing or elution of each step is carried out for 5-10 minutes at 4 ℃. The eluate was collected and its RFU value was measured.

RFU value measurement method: the RFU value of the solution sample was tested by the ultraviolet absorption method under the conditions of 488nm for excitation wavelength (Ex) and 507nm for emission wavelength (Em). Detecting parameters: for the liquid samples collected in the above process, 10 μ L of samples were taken for 3 sample detections, respectively, with the detection wavelength of Ex/Em:488nm/507nm and the detection machine of InfiniteF 200.

Drawing up a standard curve according to the purified eGFP, and converting the calculated RFU value of the eGFP into a formula of protein mass concentration, wherein the formula is as follows:

where X is the protein mass concentration (. mu.g/mL) and Y is the RFU fluorescence reading. The method for calculating the binding force of the purified glass beads comprises the following steps: substituting the numerical value of Y into the formula to obtain X which is the protein mass concentration, multiplying the X by the elution volume to obtain the eluted protein mass (W), dividing the W by the bed volume of the glass beads, and calculating to obtain the mass of the glass beads in unit volume combined with the target protein, namely the binding force, wherein the unit is mg/mL.

The binding force of the four purified glass microbeads of examples 1-4 to histidine-tagged mEGFP is shown in Table 2.

Table 2 binding of the four purified glass microbeads of examples 1-4 to histidine-tagged mmefp.

EXAMPLE 6 testing of binding

mu.L of the four eluates of example 5 were sampled and added to 8. mu.L of loading buffer (250mM pH 6.8Tris-HCl, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue (BPB, CAS:115-39-9), 50% (v/v) glycerol, 5% (v/v) beta-mercaptoethanol), mixed well, boiled for 8 minutes, and the total amount was subjected to SDS-PAGE electrophoresis. The results are shown in FIG. 5. The glass powder, the hollow glass beads 20 and the hollow glass beads 40 all have obvious single target bands (about 27kDa) and basically have no other miscellaneous bands; the hollow glass beads 60 had distinct target bands with some miscellaneous bands. Wherein, the protein purification of the

hollow glass beads

20 and 40 is over 95 percent.

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> purified glass bead, preparation method and application thereof

<130> 2020

<141> 2020-12-18

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 714

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctccttc 300

aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc 480

atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caag 714

<210> 2

<211> 238

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 2

Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

1 5 10 15

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu

20 25 30

Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

65 70 75 80

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

85 90 95

Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

130 135 140

Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly

145 150 155 160

Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 3

<211> 873

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

gcccagcacg acgaggccca gcaaaacgcc ttttaccagg ttttgaacat gcccaacttg 60

aacgccgacc agaggaacgg cttcatccag agtttgaaag acgaccccag tcaaagtgcc 120

aacgttttgg gcgaggcaca gaaattaaat gacagccagg cccccaaggc cgacgcccag 180

cagaacaact tcaacaagga tcagcagagt gccttttacg aaatattgaa catgcccaac 240

ctaaacgagg cacagagaaa tgggttcatc cagagtttaa aagatgatcc cagtcagagt 300

accaacgttt tgggggaggc caaaaagtta aacgaaagcc aggcgcccaa agctgataac 360

aacttcaaca aggagcagca gaacgccttc tacgagattc taaacatgcc caacttgaac 420

gaggagcaga ggaacggctt catccagagt ttgaaagacg atcccagtca gagtgcaaac 480

ttgctatctg aggccaagaa gctcaacgag tcacaggccc ccaaggccga taacaagttc 540

aacaaggagc agcagaacgc tttctatgaa atccttcatt tgccaaattt gaatgaggaa 600

cagaggaacg gcttcatcca gagtttgaaa gatgacccga gtcagagtgc caacttgttg 660

gccgaagcca agaagttgaa tgatgcccag gcccccaagg ccgacaacaa gtttaacaag 720

gagcagcaga atgcctttta cgagatcttg cacttgccca acttgactga ggaacaaagg 780

aacggtttca tacagagttt gaaggacgac ccctctgtta gtaaggaaat ccttgcggag 840

gcaaagaagc taaacgatgc tcaagcacca aaa 873

<210> 4

<211> 585

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

acttacaaat taatccttaa tggtaaaaca ttgaaaggcg aaacaactac tgaagctgtt 60

gatgctgcta ctgcagaaaa agtcttcaaa caatacgcta acgacaacgg tgttgacggt 120

gaatggactt acgacgatgc gactaagacc tttacagtta ctgaaaaacc agaagtgatc 180

gatgcgtctg aattaacacc agccgtgaca acttacaaac ttgttattaa tggtaaaaca 240

ttgaaaggcg aaacaactac tgaagctgtt gatgctgcta ctgcagaaaa agtcttcaaa 300

caatacgcta acgacaacgg tgttgacggt gaatggactt acgacgatgc gactaagacc 360

tttacagtta ctgaaaaacc agaagtgatc gatgcgtctg aattaacacc agccgtgaca 420

acttacaaac ttgttattaa tggtaaaaca ttgaaaggcg aaacaactac taaagcagta 480

gacgcagaaa ctgcagaaaa agccttcaaa caatacgcta acgacaacgg tgttgatggt 540

gtttggactt atgatgatgc gactaagacc tttacggtaa ctgaa 585

<210> 5

<211> 117

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 5

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn

20 25 30

Arg Tyr Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Trp Val Ala Gly Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Asn Val Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 6

<211> 423

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

atgagtgatg ttcaatcttc tttgactggt acttggtata atgaattgaa ttctaaaatg 60

gaattgactg ctaataaaga tggtactttg actggtaaat atttgtctaa agttggtgat 120

gtttacgttc catatccatt gtctggtaga tataatttgc aaccaccagc tggtcaaggt 180

gttgctttgg gttgggctgt ttcttgggaa aattctaaaa ttcattctgc tactacttgg 240

tctggtcaat tcttctctga atcttctcca gttattttga ctcaatggtt attgtcttct 300

tctactgcta gaggtgatgt ttgggaatct actttggttg gtaacgattc tttcactaaa 360

actgctccaa ctgaacaaca aattgctcat gctcaattgc attgtagagc tccaagattg 420

aaa 423

<210> 7

<211> 693

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

gtttcaaagg gtgaagctgt tattaaggag tttatgagat tcaaagtgca tatggaaggt 60

tctatgaatg gtcatgaatt tgaaattgag ggtgaaggtg aaggtagacc atatgaaggt 120

actcaaactg ctaaattgaa ggttactaaa ggtggtccat tgccattctc atgggatatt 180

ttgtcaccac aattcatgta tggttctaga gctttcatta agcatccagc tgatattcca 240

gattactata agcaatcatt cccagaaggt ttcaagtggg aaagagttat gaattttgaa 300

gatggtggtg ctgttactgt tactcaagat acttcattgg aagatggtac tttgatctat 360

aaggttaagt tgagaggtac taatttccca ccagatggtc cagttatgca aaagaaaact 420

atgggttggg aagctagtac tgaaagattg tatccagaag atggtgtttt gaagggtgac 480

attaagatgg ctttgagatt gaaagatggt ggtagatatt tggctgattt caagactact 540

tataaggcta agaagccagt tcaaatgcca ggtgcttaca atgttgatag aaaattggat 600

atcacctctc ataatgaaga ttatactgtt gttgagcaat acgaaagatc tgaaggtaga 660

cattctactg gtggtatgga tgaattgtat aag 693

Claims

1. The purified glass bead is characterized by comprising a glass bead body, wherein the glass bead body is white, a functional element is bonded on the outer surface of the glass bead body, and the functional element is used as a purification medium or can be further bonded with the purification medium; preferably, the glass bead body is a silica-coated glass bead.

2. The purified glass microspheres of claim 1, wherein the glass microsphere body has a cavity;

preferably, the average density of the glass bead body is less than 1g/cm³(ii) a More preferably, the average density of the glass bead body is 0.2-0.6 g/cm³(ii) a In a more preferred embodiment, the glass bead body has an average density of 0.4 to 0.5g/cm³(ii) a In a more preferred embodiment, the glass bead bulk has an average density of 0.4g/cm³。

3. The purified glass microspheres of any one of claims 1-2, wherein the glass microspheres have a bulk average particle size selected from any one of the following values or a range between any two of the following values: 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm;

in a preferred embodiment, the average particle size of the glass bead body is selected from 10 to 1000 μm;

in a preferred embodiment, the average particle size of the glass bead body is selected from 10 to 500 μm;

in a preferred embodiment, the average particle size of the glass bead body is selected from 10 to 250 μm;

in a preferred embodiment, the average particle size of the glass bead body is selected from 10 to 200 μm;

in a preferred embodiment, the average particle size of the glass bead body is selected from 10 to 150 μm;

in a preferred embodiment, the average particle size of the glass bead body is selected from 10 to 100 μm;

in a preferred embodiment, the glass bead has an average particle size of 20 to 100 μm;

in a preferred embodiment, the average particle size of the glass bead body is selected from 50 to 100 μm;

in a preferred embodiment, the glass beads have an average particle size of 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, with a variation of ± 20%, more preferably ± 10%.

4. The purified glass microbead according to any of claims 1-3, wherein a comb-shaped polymer is fixed on the outer surface of the glass microbead body, the comb-shaped polymer has a linear main chain and branched chains distributed along the linear main chain, the linear main chain is fixed on the outer surface of the glass microbead body, and the tail ends of the branched chains are connected with the functional element.

5. The purified glass microspheres of any one of claim 4, wherein: the comb polymer is an acrylic polymer having a-C (CO-) -C-unit structure, and the-C (CO-) -C-unit structure is one of repeating unit structures;

in one of the preferred embodiments, the monomer unit of the acrylic polymer includes one of acrylic acid, acrylate, methacrylic acid, methacrylate ester, or a combination thereof;

in one preferred mode, the linear main chain of the comb polymer is a polyolefin main chain; in a more preferred mode, the linear backbone of the comb polymer is a polyolefin backbone provided by an acrylic polymer; in one of the more preferred modes, the comb polymer is represented by-CH (CO-) -CH₂-is a repeating unit.

6. The purified glass microspheres of any one of claims 4-5, wherein the functional element is a metal ion;

preferably, the metal ion is Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof;

in one of the preferred modes, the comb polymer is represented by-CH (CO-) -CH₂-is a repeating unit, said comb polymer binding nickel ions via a pendant-CO-and a tricarboxyamino group, said pendant-CO-forming an amide bond with said tricarboxyamino group; more preferably, the tricarboxyamino group is the residue of N, N-bis (carboxymethyl) -L-lysine or nitrilotriacetic acid.

7. The purified glass microbead according to any of claims 4-5, wherein the functional element is any of biotin, biotin analogue, avidin analogue, biotin-avidin complex, biotin analogue-avidin complex, biotin-avidin analogue complex, biotin analogue-avidin analogue complex.

8. The purified glass microbeads of any one of claims 4-5, wherein purification media are bound to the ends of the branches of said comb polymer and said purification media are attached to the ends of the branches of said comb polymer by attachment elements comprising affinity complexes;

preferably, the affinity complex is selected from the group consisting of: any one of a biotin-avidin complex, a biotin analogue-avidin complex, a biotin-avidin analogue complex, and a biotin analogue-avidin analogue complex; the order between the two components of the affinity complex is an optional order;

in a more preferred embodiment, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof;

in one preferred mode, the ends of the branches of the comb polymer are bound with a purification medium, the purification medium is an affinity protein, and the purification medium is connected to the ends of the branches of the comb polymer through affinity complex interaction; 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, in which the order between the two components is optional; more preferably, the purification media is attached to the branched ends of the comb polymer in the form of a biotin-avidin protein, wherein biotin-avidin forms an affinity complex.

9. The purified glass microbead according to any of claims 1-5 and 8, wherein the purification medium contains metal ions, biotin-type tags, avidin-type tags, polypeptide-type tags, protein-type tags, immuno-type tags, or a combination thereof;

the metal ion is preferably Ca²⁺、Mg²⁺、Ni²⁺、Co²⁺Or a combination thereof;

in one of the preferred embodiments, the biotin-type tag is biotin, a biotin analogue capable of binding avidin analogue, or a combination thereof;

in one of the preferred embodiments, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof;

in a more preferable mode, a comb-shaped polymer is fixed on the outer surface of the glass bead body, and the tail end of a branched chain of the comb-shaped polymer is connected with biotin; the purification medium is avidin, and forms the binding function of an affinity complex with the biotin;

in a preferred embodiment, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof;

in a preferred embodiment, the protein-based tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably one, said affinity protein is selected from the group consisting of: protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof;

in a preferred embodiment, the immunological label is any one of an antibody-type label and an antigen-type label;

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 mode, 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 an antibody VHH fragment of an anti-protein;

in a preferred mode, the antibody type tag is an antibody scFV fragment of an anti-protein;

in one preferred mode, the antibody type tag is a nanobody against fluorescent protein;

in one preferred mode, the antibody type tag is a nano antibody against green fluorescent protein or a mutant thereof;

in a preferred embodiment, the antibody type tag is an antibody Fab fragment;

in a preferred embodiment, the antibody-type tag is an antibody F (ab') 2 fragment;

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

10. The purified glass microspheres of any one of claims 4-9, wherein: the purification medium is combined at the tail ends of the branched chains of the comb-shaped polymer, and the connection mode of the purification medium and the tail ends of the branched chains of the comb-shaped polymer is as follows: a covalent bond, a supramolecular interaction, a linking element, or a combination thereof;

in a preferred embodiment, 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 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;

11. A method for producing the purified glass microspheres as claimed in claim 4, comprising the steps of:

step two: wrapping the surface of the glass bead with a silicon dioxide coating to obtain a glass bead wrapped by silicon dioxide, namely a glass bead body;

step five: chemically modifying the tail end of a branched chain of the comb-shaped polymer, and combining with a functional element to obtain the purified glass bead;

in one preferred mode, the coating of silica on the surface of the glass bead refers to a covalent chemical modification of silica on the surface of the glass bead;

step five: chemically modifying the tail end of a branched chain of the comb-shaped polymer, and combining with a purification medium to obtain the purified glass bead;

step five: activating the tail end of a branched chain of the comb-shaped polymer, carrying out covalent reaction with tricarboxyamine, and chelating nickel ions to obtain purified glass beads, wherein the purified glass beads are nickel ion modified glass beads;

some preferred examples of the tricarboxyamine are N, N-bis (carboxymethyl) -L-lysine, nitrilotriacetic acid, and combinations thereof;

the chelating nickel ion is preferably realized in one mode, such as reaction with nickel sulfate;

step five: activating the tail ends of the branched chains of the comb-shaped polymer, and chemically modifying the tail ends of the branched chains to combine with biotin or biotin analogues to obtain the purified glass beads, wherein the purified glass beads are biotin or biotin analogue modified glass beads;

step five: activating the tail end of the branched chain of the comb-shaped polymer, and chemically modifying the tail end of the branched chain to combine with avidin or avidin analogues to obtain the purified glass beads, wherein the purified glass beads are the glass beads modified by the avidin or avidin analogues;

optionally, step six, further comprising renewing or replacing said purification medium by eluting said avidin or analog thereof under suitable conditions and then re-binding the same or a different avidin or analog thereof;

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

step six: reacting the biotin or biotin analogue modified glass beads with avidin or an analogue-purification medium covalent conjugate thereof to form an affinity complex connecting element of biotin or an analogue thereof-avidin or an analogue thereof to obtain the purified glass beads, wherein the purified glass beads are the purification medium modified glass beads;

optionally, step seven, further comprising renewing or replacing the purification media by eluting the covalent conjugates of avidin or an analog thereof with the purification media under suitable conditions and re-conjugating the same or a different covalent conjugate of avidin or an analog thereof with the purification media;

the avidin-purification medium covalent conjugates are preferably avidin-avidin covalent conjugates, in which case the purified glass beads are avidin-modified glass beads;

independently, one of the preferable modes of each step II is to add the glass beads into a mixed solvent of ethanol and water, stir, add ammonia water, and dropwise add an ethanol solution of tetraethyl orthosilicate to obtain the glass beads wrapped by silicon dioxide;

independently, one of the preferable modes of each step III is to add the glass beads wrapped by the silicon dioxide into absolute ethyl alcohol, add a silane coupling agent functionalized by carbon-carbon double bonds, mix and react to obtain the glass beads modified by the carbon-carbon double bonds; the carbon-carbon double bond functionalized silane coupling agent is preferably an acryloyl-functionalized or allyl-functionalized silane coupling agent;

more preferably, adding the glass microspheres wrapped by the silicon dioxide into absolute ethyl alcohol, adding a silane coupling agent KH570, mixing, and heating for reaction to obtain carbon-carbon double bond modified glass microspheres;

independently, one of the preferable modes of each step three is to add the glass beads wrapped by the silicon dioxide into absolute ethyl alcohol, add an aminated silane coupling agent, mix and react to obtain amino-modified glass beads; further carrying out covalent reaction with acrylic acid molecules to obtain the carbon-carbon double bond modified glass beads; more preferably, the aminated silane coupling agent is silane coupling KH 550;

independently of each other, the acrylic molecule in each of the three above steps is preferably acrylic acid, acrylate, methacrylic acid, methacrylate or a combination thereof; more preferably, the acrylic monomer molecule is sodium acrylate.

12. Use of the purified glass microbeads of any one of claims 1-10 for purifying fluorescent markers that can be bound, i.e. captured, by a purification medium carried directly by said purified glass microbeads or by a purification medium carried after modification; preferably, the fluorescent marker can be specifically combined with a purification medium directly carried by the purified glass microsphere or a purification medium carried after modification.

13. A method for purifying a fluorescent protein marker, wherein the fluorescent marker is purified by using the purified glass beads of any one of claims 1 to 10, the purified glass beads are bound with a purification medium, and the fluorescent marker can be specifically bound with the purification medium; after the purified glass beads are combined with the fluorescent marker, the color can be observed by naked eyes;