DNA microsphere as well as preparation method and application thereof

Abstract

The invention provides a DNA (deoxyribonucleic acid) microsphere which is characterized by comprising a microsphere body, a polymer connected to the surface of the microsphere body and DNA combined with the polymer. The DNA microspheres provided by the invention can be stably used in an in-vitro protein synthesis reaction, the protein preparation cost is remarkably reduced, a potential way is provided for large-scale industrial protein production, and research, development and production of protein drugs are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and specifically relates to a biological microsphere and a preparation method and application thereof. Background Art

[0002] DNA templates play a key role in in vitro gene expression systems, but their high cost and low utilization rate have always restricted the industrialization of related technologies. The synthesis of DNA templates mainly relies on chemical synthesis or bacterial amplification, which is costly and difficult to mass produce, and cannot meet the needs of industrialization. In addition, DNA templates are difficult to reuse in in vitro transcription and translation reactions, resulting in a waste of resources.

[0003] Reusable DNA templates can maximize the role of templates and realize the recycling of resources. Therefore, it is particularly necessary to develop low-cost and reusable DNA templates. The development of artificial synthetic biology has provided new ideas for the construction of this new type of template. Compared with DNA circular technology, linear template technology and other methods connect DNA templates to microspheres or other solid carriers, which can realize the separation, elution and reuse of templates. It has the advantages of simple operation, low cost and strong functional scalability.

[0004] An in vitro biosynthesis system refers to a system in which specific chemical molecules or biological macromolecules (DNA, RNA, proteins) are efficiently synthesized in vitro by adding exogenously encoded nucleic acids DNA, RNA, substrates and energy sources into a lysis system of bacteria, fungi, plant cells or animal cells.

[0005] Common in vitro biosynthesis systems include the in vitro protein synthesis system, that is, the cell-free protein synthesis system, which uses exogenous mRNA or DNA templates and cell lysates to complete the rapid and efficient translation of exogenous recombinant proteins; another example is the in vitro RNA synthesis system (IVT).

[0006] Some in vitro protein synthesis systems are in vitro transcription-translation systems (IVTT), which use DNA templates, RNA polymerase to transcribe mRNA intermediates, and then use components such as amino acids and ATP to complete the one-step efficient translation of exogenous proteins. At present, common commercial in vitro protein expression systems include Escherichia coli extract (ECE), rabbit reticulocyte lysate (RRL), wheat germ extract (WGE), insect cell extract (ICE) and human systems.

[0007] Compared with traditional in vivo recombinant expression systems, in vitro cell-free protein synthesis systems have many advantages, such as the ability to express special proteins that are toxic to cells or contain non-natural amino acids (such as D-amino acids), the ability to directly use PCR products as templates to simultaneously synthesize multiple proteins in parallel, and the ability to conduct high-throughput drug screening and proteomics research.

[0008] Existing in vitro synthesis systems have the following limitations:

[0009] 1. Existing in vitro synthesis systems all rely on mRNA or DNA templates, and the amount of template is usually much larger than the amount of protein synthesized in the end, which limits the industrial application of in vitro protein synthesis technology and the potential for cost reduction. At present, the cost of in vitro protein synthesis is still high and it is difficult to meet the needs of large-scale industrial production.

[0010] 2. Currently, the commercialized in vitro protein synthesis systems mainly include prokaryotic systems (such as E. coli) and eukaryotic systems (such as insect cells, mammalian cells, yeast, etc.). In contrast, prokaryotic systems such as E. coli are easier to culture and manipulate, with high yields, but the activity and accuracy of the synthesized proteins are lower than those of eukaryotic systems. Proteins synthesized by eukaryotic cell systems have higher activity and post-translational modifications, but are expensive and difficult to produce on a large scale.

[0011] 3. The application scope of in vitro protein synthesis technology is relatively limited, mainly concentrated in the field of drug research and development, and used to produce recombinant proteins or therapeutic protein drugs for in vivo research. The application potential in industrial production and other fields needs to be developed.

[0012] 4. At present, the mainstream technology for global industrial protein production is still based on cell fermentation. Compared with cell fermentation, in vitro protein synthesis technology has the advantages of simple operation, no risk of viral contamination and high yield, but it has high costs and low industrialization, and it is difficult to replace the dominant position of cell fermentation in industrial protein production.

[0013] In summary, although in vitro protein synthesis technology has great potential, its industrialization and large-scale application are currently at a low level. The main obstacles are high cost and strong template dependence. Breakthroughs in related technologies will help in vitro protein synthesis technology to be more widely used in protein drug research and development and industrial production.

[0014] Current status of DNA template technology:

[0015] 1. DNA templates are mainly used to guide in vitro transcription and translation reactions and synthesize RNA and proteins. Commonly used DNA templates include plasmid DNA, linear DNA, and circular DNA. These DNA templates must be obtained through bacterial transformation, PCR amplification, or synthesis.

[0016] 2. Currently, the acquisition of DNA templates mainly relies on chemical synthesis or bacterial transformation and amplification, which is costly and cumbersome to operate, making it difficult to industrialize and apply on a large scale. The cost of new-generation gene synthesis technologies, such as gene synthesis chip technology, has decreased, but the cost is still high for the demand for DNA templates for industrial production.

[0017] 3. In in vitro transcription and translation systems, in vitro reactions usually require a large amount of DNA template (exceeding the mass of the final product), which limits the application of related technologies on an industrial scale. The method of reusing or continuously using DNA templates can reduce costs and achieve industrial production.

[0018] 4. In recent years, some new methods have achieved the repeated or continuous use of DNA templates, such as DNA recycling technology, linear expression template technology, isothermal amplification, etc. The emergence of these new methods has provided new ideas for the large-scale use of DNA templates in industrial applications. However, the industrialization level of related technologies is still relatively low, and the cost advantage needs to be further improved.

[0019] Advantages and disadvantages of other DNA template reuse or serial utilization technologies:

[0020] 1. Advantages of DNA recycling technology: DNA templates can be reused continuously, reducing the demand for templates; the operation is simple and easy to implement.

[0021] Disadvantages: The design of DNA cycling strategy is relatively complex, and different templates have different cycling functions and stabilities; cyclic DNA amplification is prone to introduce errors and requires highly sensitive detection methods.

[0022] 2. Advantages of linear expression template technology: Linear DNA is easy to obtain by chemical synthesis and has low cost; it has a certain template stability and can be used continuously.

[0023] Disadvantages: Linear DNA has low stability in in vitro transcription and translation reactions and its utilization efficiency is lower than that of DNA recycling; special treatment is required to prevent linear DNA from degrading during the reaction.

[0024] 3. Advantages of isothermal amplification: The reaction operation is simple, and PCR is used to automatically amplify DNA at a constant temperature, which is highly efficient.

[0025] Disadvantages: Requires dedicated primers and DNA polymerase, which are relatively expensive; the amplified DNA sequence needs to be designed to introduce loop or enzyme cutting sites, which is relatively cumbersome; the yield of amplified DNA is difficult to accurately control and there are certain differences.

[0026] 4. Advantages of CRISPR-Cas9 gene editing technology: It can modify DNA templates in a targeted manner and achieve reuse; it is relatively easy to operate and is based on the principle of enzymatic cleavage of DNA-RNA complexes.

[0027] Disadvantages: gRNA design and synthesis are required, which is costly; it is difficult to completely avoid the production of a certain proportion of non-specific editing products. Summary of the invention

[0028] In view of the fact that the existing in vitro protein synthesis system is difficult to meet the needs of daily biological research and development and industrialization, the present invention provides a reusable DNA template that can be stably used in in vitro protein synthesis reactions, significantly reducing the cost of protein preparation, providing a potential path for large-scale industrial protein production, and facilitating the research and development and production of protein drugs.

[0029] To this end, the present application has found through research that a new DNA template microsphere can be prepared by connecting the DNA template to the microsphere, and the DNA microsphere can be used as a reusable DNA template. This solution can not only reduce the cost of DNA templates to achieve industrial production, but also open up new research directions in the field of artificial synthetic biology (cell directed evolution and functional regulation, etc.), bringing new opportunities for the widespread application of DNA template technology (gene therapy, synthetic biology research, etc.).

[0030] In particular, we selected biocompatible microspheres as solid carriers, linked linear template DNA with chemical bonds, and optimized reaction conditions to achieve high connection efficiency and template stability. We also verified its stability in the reaction, the number of reuses and efficiency and other technical indicators, indicating that this new DNA microsphere has great application potential in industrial production and reducing the cost of gene expression.

[0031] The first aspect of the present invention provides a DNA microsphere, characterized in that it comprises: a microsphere body, a polymer connected to the surface of the microsphere body, and DNA bound to the polymer.

[0032] Further preferably, the polymer binds to DNA via a functional group.

[0033] Further preferably, the functional group is selected from carboxyl, hydroxyl, amino, thiol, a salt form of carboxyl, a salt form of amino, an ester group, or a combination of the aforementioned functional groups.

[0034] Further preferably, the DNA is modified by the functional group.

[0035] Further preferably, the DNA is obtained by amplification of a DNA primer sequence.

[0036] Further preferably, the length of the DNA primer sequence is 6 to 30 bp; preferably 9 to 21 bp.

[0037] Further preferably, the amplification is performed after the DNA primer sequence is combined with the polymer via the functional group.

[0038] Further preferably, the DNA primer sequence is combined with the polymer via the functional group to perform the amplification and then connected to the surface of the microsphere body to obtain the DNA microsphere.

[0039] More preferably, the polymer has branched chains.

[0040] Further preferably, the polymer binds to DNA via a branched chain.

[0041] Further preferably, the polymer is directly or indirectly connected to the outer surface of the microsphere body through a connecting group.

[0042] More preferably, one end of the linking group is connected to the microsphere, and the other end is connected to the polymer by combining with an active group (such as an amino group, a carboxyl group, etc.) at the end of the polymer.

[0043] Further preferably, the outer surface of the microsphere body has at least one linear polymer with branches, one end of the linear polymer with branches is connected to the outer surface of the microsphere body, and the other part is free from the outer surface of the microsphere body; the branch of the linear polymer is connected to DNA.

[0044] Further preferably, the linear polymer is fixed to the outer surface of the microsphere body directly or indirectly through a connecting group.

[0045] Further preferably, the linking group is selected from one or a combination of substituted or unsubstituted siloxy groups, amide groups, ester groups, carbonyl groups, alkenyl groups, alkynyl groups, alkylene groups, ether groups, and thioether groups.

[0046] More preferably, the surface of the microsphere body contains silicon or silicon oxide.

[0047] Further preferably, the material of the microsphere body is selected from glass, ceramic or magnetic material.

[0048] Further preferably, the microsphere body is a magnetic microsphere.

[0049] Further preferably, the microspheres are selected from glass, ceramic or magnetic materials.

[0050] Further preferably, the chemical composition of the magnetic material includes any one of iron compounds, iron alloys, zinc oxides, manganese oxides, gadolinium oxides, cobalt compounds, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides, chromium oxides or any combination thereof.

[0051] Further preferably, the chemical composition of the magnetic material includes any one of iron oxide, zinc oxide, manganese oxide, gadolinium oxide, cobalt oxide, nickel oxide, manganese oxide, zinc oxide, gadolinium oxide, chromium oxide or any combination thereof.

[0052] Further preferably, the chemical composition of the magnetic material includes iron oxide, iron, cobalt, Co 2+ , iron nitride, Mn3O4, GdO, nickel, Ni 2+ Any one or any combination thereof; wherein the iron oxide is preferably Fe3O4, γ-Fe2O3 or a combination thereof.

[0053] Further preferably, the chemical composition of the magnetic material includes any one of Fe3O4, γ-Fe2O3, iron nitride, Mn3O4, AlNi(Co), FeCr(Co), FeCrMo, FeAlC, AlNi(Co), FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNi(Mo), FeSi, FeAl, FeNi(Mo), FeSiAl, BaO·6Fe2O3, SrO·6Fe2O3, PbO·6Fe2O3, GdO or any combination thereof.

[0054] Further preferably, the microspheres are selected from glass microspheres, ceramic microspheres or glass-ceramic microspheres.

[0055] Further preferably, the microspheres may be hollow.

[0056] Further preferably, the size of the microsphere body is selected from any one of the following particle sizes or the range between any two particle sizes: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, , 8.5μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm; the diameter sizes are average values.

[0057] More preferably, the diameter of the microsphere body is selected from 0.1 to 10 μm.

[0058] More preferably, the diameter of the microsphere body is selected from 0.2 to 6 μm.

[0059] More preferably, the diameter of the microsphere body is selected from 0.4 to 5 μm.

[0060] More preferably, the diameter of the microsphere body is selected from 0.5 to 3 μm.

[0061] More preferably, the diameter of the microsphere body is selected from 0.2 to 1 μm.

[0062] More preferably, the diameter of the microsphere body is selected from 0.5 to 1 μm.

[0063] Further preferably, the average diameter of the microsphere body is 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, with a deviation of ±20%, more preferably ±10%.

[0064] Further preferably, the main chain of the linear polymer is a polyolefin main chain or an acrylic polymer main chain.

[0065] Further preferably, the linear backbone of the polymer is a polyolefin backbone and is provided by a backbone of an acrylic polymer.

[0066] Preferably, the average molecular weight MW of the polymer is 1000-5000000; more preferably, the MW is 1000-100000; further preferably, the MW is 2000-50000; further preferably, the MW is 3000-10000.

[0067] More preferably, the monomer unit of the acrylic polymer is one of acrylic acid, acrylic acid salt, acrylic acid ester, methacrylic acid, methacrylic acid salt, methacrylic acid ester, or a combination thereof.

[0068] The second aspect of the present invention provides a method for preparing the DNA microspheres described in the first aspect of the present invention.

[0069] Further preferably, the preparation method comprises the following steps: (1) chemically modifying the microsphere body by using an amino silane coupling agent to introduce amino groups into the outer surface of the microsphere body to form amino-modified microsphere A; (2) modifying the DNA primer sequence to obtain a modified DNA primer sequence; (3) combining the modified DNA primer sequence with a polymer and then amplifying it to obtain polymer-modified DNA; (4) reacting the polymer-modified DNA with the amino-modified microsphere A to obtain the DNA microsphere.

[0070] Further preferably, the amino silane coupling agent is selected from Wherein R5 to R7 are each independently selected from C1-C 10 The alkyl group is an alkyl group, and m is 1-10.

[0071] More preferably, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0072] More preferably, R5 to R7 are each independently selected from an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0073] Further preferably, the amino silane coupling agent is selected from 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane.

[0074] Further preferably, in step (2), the 5' end of the DNA primer sequence is modified with the amino group; more preferably, the amino group is modified at the C2-C20 position of the 5' end; further preferably, the amino group is modified at the C5-C10 position of the 5' end; most preferably, the amino group is modified at the C6 position of the 5' end.

[0075] Further preferably, the length of the DNA primer sequence is 6 to 30 bp; preferably 9 to 21 bp.

[0076] Further preferably, the DNA primer sequence is GGCGTAGAGGAT.

[0077] Further preferably, the amplification is performed after the DNA primer sequence is combined with the polymer via the functional group.

[0078] Further preferably, the DNA primer sequence is combined with the polymer via the functional group to perform the amplification and then connected to the surface of the microsphere body to obtain the DNA microsphere.

[0079] Further preferably, the modification in step (2) is to react the DNA sequence with a polyolefin polymer.

[0080] Further preferably, the polyolefin polymer is selected from one or more of substituted or unsubstituted polypropylene, polyacrylic acid, polyacrylic acid salt, polyacrylic acid ester or polyacrylamide.

[0081] More preferably, the substituent is selected from C1-C20 alkyl groups.

[0082] Further preferably, the polyolefin polymer is selected from polyacrylic acid, polyacrylic acid salt, polymethacrylic acid or polymethacrylate salt.

[0083] Further preferably, in the step (4), before reacting the polymer-modified DNA with the microsphere A, the system containing the microsphere A is first subjected to ultrasonic vibration.

[0084] More preferably, the ultrasonic oscillation time is 1 to 30 min, more preferably 5 to 20 min, and particularly preferably 5 to 10 min.

[0085] The third aspect of the present invention provides the use of the DNA microspheres described in the first aspect of the present invention.

[0086] Further preferably, the DNA microspheres can be used as DNA templates.

[0087] More preferably, the DNA template is a DNA template of a target protein (or exogenous protein).

[0088] Further preferably, the target protein is selected from the following group: luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, cytokine, interferon α2b, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine carrier protein, tyrosinase, xylanase, or a combination thereof.

[0089] The fourth aspect of the present invention provides the use of the DNA microspheres described in the first aspect of the present invention as DNA templates in RNA transcription or protein synthesis.

[0090] Further preferably, the RNA transcription or protein synthesis is preferably in vitro RNA transcription or in vitro protein synthesis.

[0091] The fifth aspect of the present invention provides a cell-free in vitro synthesis system, comprising: (1) cell extract; and (2) the DNA microspheres described in the first aspect of the present invention.

[0092] Further preferably, the cell-free in vitro synthesis system is a cell-free protein synthesis system.

[0093] Further preferably, the cell extract is derived from eukaryotic cells or prokaryotic cells.

[0094] Further preferably, the eukaryotic cell source includes but is not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; the mammalian cell source may include but is not limited to mouse source (including rats, mice, guinea pigs, golden hamsters, hamsters, etc.), rabbit source, monkey source, human source, pig source, sheep source, cow source, dog source, horse source, etc.

[0095] Further preferably, the yeast cell is selected from Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia aminuta, Ogataeaminuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia alictaria, Pichia g uercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia asp., Saccharomyces cerevisiae. cerevisiae), brewer's yeast, sugar cane molasses yeast, Saccharomyces cerevisiae, Hansenula polymorpha, Candida utilis, Kluyveromyces cerevisiae, or a combination thereof.

[0096] Further preferably, the Kluyveromyces further includes: the Kluyveromyces includes: Kluyveromyces lactis (Kluyveromyces, K.lactis), Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or a combination thereof.

[0097] The sixth aspect of the present invention provides a cell-free in vitro synthesis method, which is prepared using the system provided by the fifth aspect of the present invention.

[0098] Further preferably, the cell-free in vitro synthesis method is a cell-free protein synthesis method.

[0099] Compared with the prior art, the present invention has the following beneficial effects:

[0100] (1) Higher stability: Compared with free DNA templates, the DNA template attached to the microsphere surface is more stable, degrades more slowly under in vitro reaction conditions, has a longer reaction life and a higher number of utilizations.

[0101] (2) The template concentration can be precisely controlled: the concentration of template DNA can be precisely controlled by controlling the microsphere loading capacity to meet the needs of different reactions.

[0102] (3) Low cost: Compared with PCR and gene editing technology, the cost of obtaining microsphere-linked DNA templates is lower and easy to achieve industrial production.

[0103] (4) Avoid product contamination: Compared with free DNA templates, connected DNA templates can be more easily removed from the reaction products by elution, reducing DNA contamination of the products. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The activity data of DNA magnetic microspheres prepared in Examples 1-3 of the present application after IVTT reaction, 75 μL magnetic beads were tested for the activity of DNA reaction after treatment with 200 μL activation solution at room temperature (15°C), where MAG-1 to MAG-6 groups used exactly the same pre-treatment method and reaction conditions, in order to verify the repeatability and stability of the reaction activity. PC is the activity of DNA not linked to the magnetic microspheres.

[0105] Figure 2 The activity data of the supernatant corresponding to the DNA magnetic microspheres prepared in Examples 1-3 of this application after IVTT reaction, in which the MAG-1-S~MAG-6-S groups used exactly the same pre-treatment method and reaction conditions, the purpose is to verify the repeatability and stability of the reaction activity. PC is the activity of DNA not linked to the magnetic microspheres.

[0106] Figure 3The activity of the magnetic microspheres after repeated use is compared with that of the first use, as a verification of the effectiveness of the reusable DNA template. Mag 1st refers to the activity of the DNA magnetic microspheres used for the first time, mag 2nd refers to the activity of the DNA magnetic microspheres used after washing (i.e. the second time), and 1, 2, 3 refer to three parallel experiments.

[0107] Figure 4 This is a 6-fold expansion of the magnetic microsphere reaction activity verification, where MAG 6X is a 1μm magnetic microsphere that is proportionally amplified 6 times, 1, 2, and 3 are parallel experiments with exactly the same conditions, -S is the supernatant of the corresponding group, and PC is the control. (Proportional amplification of 6 times means that the amount of related magnetic microspheres, activation solution, and DNA added is amplified 6 times, which is the overall amplification of the experiment, and the particle size and concentration of the magnetic microspheres themselves remain unchanged).

[0108] Figure 5 Activity data of DNA magnetic microspheres and their supernatants prepared in Example 5 of the present application after IVTT reaction, wherein F refers to the forward primer (GGATAGCGA, 5' end C6NH2), and R refers to the reverse primer (GCGGTGGCA, 5' end C6NH2). As shown in the figure, 1F-supernatant refers to the first verification experiment of the DNA magnetic microsphere supernatant obtained by the forward primer, and 1F-beads refers to the first verification experiment of the DNA magnetic microspheres obtained by the forward primer. 1R-supernatant refers to the first verification experiment of the DNA magnetic microsphere supernatant obtained by the reverse primer, and 1R-beads refers to the first verification experiment of the DNA magnetic microspheres obtained by the reverse primer. The numbers in the figure represent the serial numbers of parallel reactions using exactly the same pre-treatment method and reaction conditions.

[0109] Figure 6 The activity data of the DNA microspheres prepared in Examples 8-10 of the present application after IVTT reaction, 100 μL of glass beads at room temperature (15°C) and 200 μL of DNA reaction after activation solution treatment, where 320 represents the mesh size of the glass beads (45 μm), and groups 1, 2, and 3 use exactly the same pretreatment method and reaction conditions to verify the repeatability and stability of the reaction activity. PC is the activity of DNA not linked to the glass microspheres.

[0110] Figure 7The activity data of the supernatant corresponding to the DNA microspheres prepared in Example 12 of the present application after IVTT reaction are used to verify the effect of ultrasonic vibration on the activity of glass beads, wherein 320 and 2000 are the mesh sizes of the glass beads (corresponding to particle sizes of 45 μm and 7.5 μm), and -1 and -2 are completely identical pretreatment methods and reaction conditions, the purpose of which is to verify the repeatability and stability of the reaction activity. U is the corresponding group that was first subjected to ultrasonic vibration for 10 minutes before the reaction. DETAILED DESCRIPTION

[0111] The present invention is further described below in conjunction with the specific embodiments and examples, and please refer to the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably followed and referred to the conditions indicated in the specific embodiments described above, and then can be followed according to conventional conditions, such as the experimental conditions described in documents such as "Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-free Protein Synthesis Experimental Manual" "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008", or according to the conditions recommended by the manufacturer.

[0112] Unless otherwise specified, the percentages and parts mentioned in the present invention are percentages and parts by weight.

[0113] Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

[0114] Unless otherwise specified, the temperature units in this application are all degrees Celsius (°C).

[0115] Nouns and terms

[0116] The following is an explanation or description of the meaning of some relevant "nouns" and "terms" used in the present invention, so as to better understand the present invention. The corresponding explanation or description applies to the full text of the present invention, both below and above. When the present invention involves references, the definitions of relevant terms, nouns, and phrases in the references are also quoted, but when they conflict with the definitions in the present invention, the definitions in the present invention shall prevail. When the definitions in the references conflict with the definitions in the present invention, it does not affect the cited components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., which shall be subject to the contents determined in the references.

[0117] In the description of the present invention, preferred modes such as "one of the preferred embodiments", "one of the preferred modes", "one of the preferred embodiments", "one of the preferred examples", "preferred examples", "in a preferred embodiment", "some preferred examples", "some preferred modes", "preferably", "preferably", "preferably", "more preferably", "more preferably", "further preferably", "most preferably", and illustrative enumeration modes such as "one of the embodiments", "one of the modes", "example", "specific example", "for example", "as an example", "for example", "such as", "such as", etc., do not constitute any limitation on the scope of coverage and protection scope of the invention, and the specific features described in each mode are included in at least one specific embodiment of the present invention. In the present invention, the specific features described in each mode may be combined in a suitable manner in any one or more specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred mode may also be combined in any suitable manner.

[0118] In the present invention, "any combination thereof" means "greater than 1" in terms of quantity, and means a group consisting of the following situations in terms of coverage: "select any one of them, or select a group consisting of at least two of them".

[0119] In the present invention, the descriptions of "one or more", "one or more" and "one or more" have the same meaning as "at least one", "at least one", "a combination thereof", "or a combination thereof", "and combinations thereof", "or any combination thereof", "and any combination thereof", etc. and can be used interchangeably to indicate that the quantity is equal to "1" or "greater than 1".

[0120] In the present invention, "or / and" and "and / or" are used to represent "optionally one of them or optionally a combination of them", and also represent at least one of them.

[0121] The prior art means described in the present invention in such ways as “usually”, “conventional”, “general”, “frequently” and “often” are also cited as references to the contents of the present invention. Unless otherwise specified, they can be regarded as one of the preferred ways of some technical features of the present invention. It should be noted that they do not constitute any limitation on the scope of coverage and protection scope of the invention.

[0122] All documents mentioned in the present invention and documents directly or indirectly cited by these documents are cited in this application as references, just as if each document was cited individually as a reference.

[0123] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (including but not limited to the embodiments) can be combined with each other to form a new or preferred technical solution, as long as it can be used to implement the present invention. Due to space limitations, they will not be described one by one.

[0124] The polymer in the present invention broadly includes oligomers and high polymers, having at least three structural units or a molecular weight of at least 500 Da (the molecular weight can be characterized by a suitable method, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.).

[0125] Acrylic monomer molecules: Monomer molecules that can be used to synthesize acrylic polymers, have the basic structure of C(COO-)=C, for example, CH(COOH)=CH2, CH(COONa)=CH2, CH3C(COOH)=CH2, CH3C(COONa)=CH2, CH(COOCH3)=CH2, CH(COOCH2CH2OH)=CH2, CH3C(COOCH3)=CH2, CH3C(COOCH2CH2OH)=CH2, etc.

[0126] The "fixed" methods such as fixed, fixed to, fixed with, fixed in, etc. refer to covalent bonding methods.

[0127] There is no particular limitation on the “connection” / “binding” methods such as carrying, connected with, connected to, connected in, bound to, captured, captured, etc., including but not limited to covalent methods, non-covalent methods, etc.

[0128] Covalent method: a method of direct bonding by covalent bonds. The covalent method includes but is not limited to a dynamic covalent method, which refers to a method of direct bonding by dynamic covalent bonds.

[0129] Covalent bonds: include common covalent bonds such as amide bonds and ester bonds, as well as dynamic covalent bonds with reversible properties.

[0130] The covalent bond includes a dynamic covalent bond. A dynamic covalent bond is a reversible chemical bond, including but not limited to an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof. Those skilled in the art of chemistry can understand its meaning.

[0131] The "modified" products, including but not limited to the derivatives, modified products, genetically modified products, fusion products, etc. of the present invention, may maintain the original functions or performances, or optimize or change their functions or performances.

[0132] In vitro protein synthesis reaction refers to the reaction of synthesizing protein in an in vitro cell-free synthesis system, including at least the translation process. Including but not limited to IVT reaction (in vitro transcription reaction), IVTT reaction (in vitro transcription and translation reaction), IVDTT reaction (in vitro replication transcription and translation reaction). In the present invention, IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as D2P system, D-to-P system, D_to_P system, DNA-to-Protein system; the corresponding in vitro protein synthesis method is also called D2P method, D-to-P method, D_to_P method, DNA-to-Protein method.

[0133] "Cell-free system" refers to the in vitro biosynthesis, which is not secretory expression by intact cells. It should be noted that in the in vitro synthesis system of the present invention, it is also allowed to add cell components to promote the reaction, but the added cells are not mainly for the purpose of secreting and expressing the exogenous target protein.

[0134] In addition, in the intact cell-free D2P system constructed under the guidance of the present invention, a small amount of intact cells are intentionally added (for example, the protein content provided by them is not more than 30wt% compared with the protein content provided by the cell extract). This "avoidance" method is also included in the scope of protection of the present invention.

[0135] Exogenous protein: The target expression product of the in vitro protein synthesis system of the present invention is not synthesized by host cell secretion, but is synthesized in vitro based on an exogenous nucleic acid template, and therefore can also be called a target protein. The exogenous protein can be a protein, a fusion protein, a mixture of protein molecules or fusion protein molecules; it also broadly includes polypeptides. The product obtained after an in vitro protein synthesis reaction based on a nucleic acid template encoding an exogenous protein can be a single substance or a combination of two or more substances. "Exogenous protein", "target protein", "target protein", "target translation product" have the same meaning and can be translated as "objective protein", "interested protein", "objective translated product", "interested protein product", etc., and can be used interchangeably in the present invention.

[0136] D2P, DNA-to-Protein, from DNA template to protein product. For example, D2P technology, D2P system, D2P method, D2P kit, etc.

[0137] "The expression system of the present invention", "the in vitro expression system of the present invention", "the in vitro cell-free expression system", and "the in vitro cell-free expression system" can be used interchangeably, all referring to the in vitro expression system of the present invention, and other descriptions can also be used, such as: protein in vitro synthesis system, in vitro protein synthesis system, cell-free system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system) and other descriptions. According to the reaction mechanism, it can include an in vitro transcription system (which can be abbreviated as an IVT system, a mR2P system), an in vitro transcription and translation system (which can be abbreviated as an IVTT system, a D2P system), an in vitro replication transcription and translation system (which can be abbreviated as an IVDTT system, a D2P system), etc. In the present invention, the IVTT system is preferred. We also call the in vitro protein synthesis system a "protein synthesis factory" ("Protein Factory" or "proteinfactory" or "Proteinfactory"). The in vitro protein synthesis system provided by the present invention adopts an open description method for its components. The cell-free protein synthesis system of the present invention uses exogenous DNA, mRNA or a combination thereof as a nucleic acid template for protein synthesis, and achieves in vitro synthesis of the target protein by artificially controlling the addition of substrates and transcription, translation-related protein factors and other substances required for protein synthesis.

[0138] One of the specific embodiments of the in vitro protein synthesis system, further includes but is not limited to, for example, the cell-free protein synthesis system based on Escherichia coli described in WO2016005982A1. Other cited documents of the present invention, including but not limited to the in vitro cell-free protein synthesis system based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus recorded in the documents directly and indirectly cited in the documents, are also included in the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45" includes but is not limited to the in vitro cell-free protein synthesis system (In vitro cell-free protein synthesis system) recorded in the cited documents on pages 27-28 of the "2.1 Systems and Advantages" section, which can be used as the in vitro protein synthesis system for implementing the present invention.For example (unless they conflict with the present invention, the following documents and their citations are cited in full content and for all purposes), documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN10942350 9A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN1100 93284A, CN110408635A, CN110408636A, CN110551745 A. CN110551700A, CN110551785A, CN110819647A, CN110845622, CN110938649A, CN110964736A, CN111378706 A. CN111378707A, CN111378708A, CN111718419A, CN111748569A, CN2019107298813, CN2019112066163, CN20 The in vitro cell-free protein synthesis systems, DNA template construction and amplification methods described in 18112862093, CN2019114181518, CN2020100693833, CN2020101796894, CN202010269333X, CN2020102693382, CN2020113574616 and the references therein can all be used as the in vitro protein synthesis systems and DNA template construction and amplification methods for implementing the present invention.

[0139] In the present invention, "protein" and "protein" have the same meaning, are both translated as protein, and can be used interchangeably.

[0140] In the present invention, "system" and "system" are both translated as system and can be used interchangeably.

[0141] In the present invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably.

[0142] In the present invention, cell extract, cell extract, cell lysate, cell disruption product and cell lysate have the same meaning and can be used interchangeably. In English, cell extract, cell lysate and the like can be used.

[0143] In the present invention, energy system, energy system, and energy supply system have the same meaning and can be used interchangeably. Energy regeneration system and energy regeneration system have the same meaning and can be used interchangeably. The energy regeneration system is a preferred embodiment or component of the energy system.

[0144] The in vitro synthesis system of the present invention further comprises one or more components selected from the group consisting of a substrate for synthesizing proteins, a substrate for synthesizing RNA, RNA polymerase, magnesium ions, potassium ions, a buffer, an energy regeneration system, polyethylene glycol (PEG) or its analogs, dithiothreitol (DTT), and an optional solvent, wherein the solvent is water or an aqueous solvent. Further, the cell extract does not contain endogenous long-chain nucleic acid molecules of yeast.

[0145] Furthermore, the substrate for synthesizing RNA includes: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.

[0146] Furthermore, the substrates for synthesizing proteins include 20 natural amino acids and unnatural amino acids.

[0147] Furthermore, the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following group: magnesium acetate, magnesium glutamate, or a combination thereof.

[0148] Furthermore, the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following group: potassium acetate, potassium glutamate, or a combination thereof.

[0149] Furthermore, the energy regeneration system is selected from the following group: a creatine phosphate / creatine phosphate enzyme system, one of the glycolysis pathway intermediate energy systems, sucrose or a combination thereof.

[0150] Furthermore, the buffer is selected from the following group: 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0151] Further, the protein synthesis system contains polyethylene glycol (PEG) or its analogs. The concentration of polyethylene glycol or its analogs is not particularly limited. Generally, the concentration (w / v) of polyethylene glycol or its analogs is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEG is selected from the following group: one of PEG3000, PEG3350, PEG6000, PEG8000 or a combination thereof.

[0152] Furthermore, the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc. Preferably, the polyethylene glycol has a molecular weight of 3000-10000.

[0153] An optional scheme is that the protein synthesis system provided by the present invention includes: yeast cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, RNA polymerase, polyethylene glycol, and sucrose.

[0154] In the present invention, the cell extract does not contain intact cells, and a typical cell extract includes ribosomes, transfer RNA, aminoacyl tRNA synthetase, initiation factors and elongation factors required for protein synthesis, and termination release factors for protein translation. In addition, the cell extract also contains some other proteins derived from the cytoplasm of the cell, especially soluble proteins.

[0155] In the present invention, the protein content of the cell extract is 20-100 mg / ml, preferably 50-100 mg / ml. The method for determining the protein content is the Coomassie Brilliant Blue determination method.

[0156] In the present invention, the preparation method of the cell extract or cell lysate is not limited. A preferred preparation method comprises the following steps:

[0157] (i) providing cells;

[0158] (ii) washing the cells to obtain washed cells;

[0159] (iii) subjecting the washed cells to cell disruption to obtain a crude cell extract;

[0160] (iv) subjecting the crude cell extract to solid-liquid separation to obtain a liquid portion, namely the cell extract.

[0161] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0162] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000×g, preferably 8000-30000×g.

[0163] In the present invention, the centrifugation time is not particularly limited, and a preferred centrifugation time is 0.5 min-2 h, preferably 20 min-50 min.

[0164] In the present invention, the centrifugation temperature is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably, at 2-6°C.

[0165] In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing liquid at a pH of 7-8 (preferably 7.4). The washing liquid is not particularly limited. Typically, the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0166] In the present invention, the cell disruption treatment method is not particularly limited. A preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.

[0167] The nucleoside triphosphate mixture in the protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate. In the present invention, the concentration of various mononucleotides is not particularly limited, and the concentration of each mononucleotide is usually 0.5-5mM, preferably 1.0-2.0mM.

[0168] The amino acid mixture in the protein synthesis system may include natural or non-natural amino acids, and may include D-type or L-type amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine. The concentration of each amino acid is generally 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.

[0169] In a preferred embodiment, the in vitro cell-free protein synthesis system further contains sucrose, and the concentration of the sucrose is 0.03-40wt%, preferably 0.08-10wt%, and more preferably 0.1-5wt%, based on the total weight of the protein synthesis system.

[0170] A particularly preferred in vitro cell-free protein synthesis system contains the following components in addition to yeast cell extract: 22 mM 4-hydroxyethylpiperazineethanesulfonic acid at pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, and 0.027-0.054 mg / mL T7 RNA polymerase.

[0171] Cell extracts

[0172] In the present invention, cell extract and cell lysate have the same meaning, and both are substances obtained after cell disruption.

[0173] Example 1 Modification of magnetic microspheres

[0174] Weigh 1-150 g of silicon dioxide-coated ferroferric oxide magnetic microspheres, wash the magnetic microspheres with anhydrous ethanol for 2-5 times, add 5-500 mL of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) ethanol solution (5%-20%, v / v) to the washed magnetic microspheres, react for 12-72 hours, and then wash the magnetic microspheres with anhydrous ethanol and distilled water for 3-5 times to obtain amino-modified magnetic microspheres A.

[0175] Example 2: Preparation of DNA samples

[0176] (1) Polyacrylic acid solution a configuration

[0177] a. Take 4 ml of polyacrylic acid solution (molecular weight 3000, mass fraction 50%), add ddH2O to 40 mL, add 1 mol / L NaOH to adjust the pH to 7.5, and add pure water to 50 mL;

[0178] b. Take 230 μL of the solution into a test tube, add ddH2O to 10 mL, and shake thoroughly.

[0179] (2) Add 318 μL of polyacrylic acid solution to a DNA primer (specific sequence: GGCGTAGAGGAT, amino modified at the C6 position of the 5' end during synthesis), shake well on a vortex for 5 min, and let stand for 25 min.

[0180] (3) Use the modified DNA primer sequence (DNA primer sequence modified by polyacrylic acid) to amplify (Ampi) to obtain the green fluorescent protein DNA sample (DNA template).

[0181] Example 3: Preparation of DNA magnetic microspheres.

[0182] (1) Take 75 μL of magnetic microspheres A (particle size 1 μm, containing liquid) and transfer them to a 2 mL centrifuge tube a.

[0183] (2) Take 200 μL of DNA sample and transfer it to another centrifuge tube b.

[0184] (3) Prepare activation solution: weigh 0.300 g of EDC and 0.150 g of NHS into a 15 mL centrifuge tube, add ddH2O to make up to 10 ml, and shake thoroughly to dissolve the drugs completely.

[0185] (4) Transfer 100 μL of activation solution to centrifuge tube b, shake on a vortex machine for 1 min, and let stand for 15 min.

[0186] (5) Transfer all the mixed liquid in centrifuge tube b to centrifuge tube a and shake centrifuge tube a manually to fully mix the contents.

[0187] (6) Place centrifuge tube a on a rotator and react at room temperature (preferably 18°C) for 24 hours to obtain a solution containing DNA magnetic microspheres.

[0188] Example 4: Activity Verification

[0189] (1) Remove centrifuge tube a and place it on a magnetic test tube rack to wait for the DNA magnetic microspheres to separate from the liquid.

[0190] (2) Transfer the supernatant to centrifuge tube c.

[0191] (3) Add ddH2O to centrifuge tube a for cleaning and aspirate the supernatant.

[0192] (4) Centrifuge tube a and centrifuge tube c are sent for inspection, and the target protein is synthesized in the IVTT system.

[0193] The IVTT system includes the following components (final concentration): 9.78 mM Tris-HCl, pH 8.0, 80 mM potassium acetate, 5 mM magnesium acetate, 1.8 mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate, each nucleoside triphosphate concentration is 1.8 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan) Acid, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each amino acid is 0.1mM), 15mM glucose, 320mM maltodextrin (molar concentration measured in glucose units, corresponding to about 52mg / mL), 24mM tripotassium phosphate, 2% polyethylene glycol 8000, and finally 50% volume of cell extract (specifically yeast cell extract, more specifically Kluyveromyces lactis cell extract) is added.

[0194] Wherein, the lactic acid Kluyveromyces extract includes endogenously expressed T7 RNA polymerase. The lactic acid Kluyveromyces extract is transformed in the following manner: using a modified strain based on the lactic acid Kluyveromyces strain ATCC8585; using the method described in CN109423496A, the coding gene of T7 RNA polymerase is integrated into the genome of lactic acid Kluyveromyces to obtain a modified strain so that it can endogenously express T7 RNA polymerase; using the modified strain to culture cell raw materials, and then preparing cell extracts. The preparation process of the lactic acid Kluyveromyces cell extract adopts conventional technical means, and is prepared by reference to the method described in CN109593656A. In summary, the preparation steps include: providing an appropriate amount of raw materials of lactic acid Kluyveromyces cells cultured by fermentation, quick freezing the cells with liquid nitrogen, breaking the cells, and centrifuging and collecting the supernatant to obtain a cell extract. The protein concentration in the obtained lactic acid Kluyveromyces cell extract is 20-40 mg / mL.

[0195] IVTT reaction: Add the components of the above centrifuge tube a and centrifuge tube c to the above in vitro protein synthesis system to perform in vitro protein synthesis reaction. Synthesize protein to obtain IVTT reaction solution containing the protein. Fluorescent protein activity assay: Place in Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescent signal, and use the relative fluorescence unit (RFU) as the activity unit (see Figure 1-2 ).

[0196] pass Figure 1-2 It can be seen that there is very little free template DNA in the centrifuge and the activity is almost negligible, indicating that the template DNA is almost completely linked to the magnetic microspheres, and the obtained DNA magnetic microspheres have high biological activity. In addition, from the activity data of MAG-1 to MAG-6, it can be seen that the activities of the six groups of magnetic beads with exactly the same reaction conditions are RFU4607, RFU4587, RFU4332, RFU4418, RFU4695, and RFU4652, which all exceed the control group PC (MAG-3 is the lowest, 102.07% of the PC activity, and MAG-5 is the highest, 110.63% of the PC activity) and the maximum activity difference between the groups is RFU363 (8.38%). The activity requirements are met. It shows that the DNA magnetic microspheres can maintain high activity as template DNA even after repeated use.

[0197] Example 5: Repeated testing of DNA magnetic microspheres after washing

[0198] After washing the DNA magnetic microspheres in the above embodiment, IVTT activity test was performed again and parallel test was performed. The test conditions were the same as those in Example 4. The results of three parallel tests showed that the activity of the magnetic beads (RFU5396-RFU5845) after washing and reuse was 98.83%-107.05% of the activity of the first use (RFU5460). The maximum decay rate was 1.17%. Assuming that the activity decreased by 1.17% each time, the activity after 20 repetitions should be 79.03%. When the PC activity was RFU5460, its activity should be RFU4315, which still met the activity requirements (see Figure 3 ).

[0199] Example 6 Expanded Experimental Verification

[0200] The amount of magnetic microspheres A, activation solution, and DNA added was increased by 6 times, and the above experiment was repeated for verification. Figure 4 As shown, the activities of MAG 6X 1, 2, and 3 groups were RFU3366, RFU3239, and RFU3610, respectively, all of which were above 100% of the PC control group, with the highest being 118% of the PC activity of MAG 6X 3 and the lowest being 105.8% of the PC activity of MAG 6X 2. This proves that even if the reaction conditions are expanded several times, the DNA sequences are almost all linked to the microspheres, and the DNA magnetic microspheres still have high activity.

[0201] Example 7 (Comparative Example): The DNA primer sequence was linked to the magnetic microspheres for re-amplification, and the activity of the magnetic microspheres was verified.

[0202] (1) Take 75 μL of magnetic microspheres A (particle size 1 μm, containing liquid) and transfer them to a 2 mL centrifuge tube a.

[0203] (2) Take 200 μL of amino-modified DNA primer sequence into another centrifuge tube b.

[0204] (3) Prepare activation solution: weigh 0.300 g of EDC and 0.150 g of NHS into a 15 mL centrifuge tube, add ddH2O to make up to 10 ml, and shake thoroughly to dissolve the drugs completely.

[0205] (4) Transfer 100 μL of activation solution to centrifuge tube b, shake on a vortex for 1 min, and let stand for 15 min.

[0206] (5) Transfer all the mixed liquid in centrifuge tube b to centrifuge tube a and shake centrifuge tube a manually to fully mix the contents.

[0207] (6) Place centrifuge tube a on a rotator and react at room temperature for 24 hours.

[0208] (7) Remove centrifuge tube a and place it on a magnetic test tube rack to wait for the magnetic beads to separate from the liquid.

[0209] (8) Take the supernatant and put it into centrifuge tube c.

[0210] (9) Add ddH2O to centrifuge tube a and wash three times, then aspirate the supernatant.

[0211] (10) The magnetic beads and DNA sequence linkers in centrifuge tube a and the free DNA sequence in centrifuge tube c are sent for amplification (green fluorescent protein DNA template).

[0212] (11) After amplification, the samples in centrifuge tubes a and c were subjected to fluorescent protein activity assay (the method is the same as in Example 4):

[0213] The samples were placed in Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescence signal, and the relative fluorescence unit (RFU) was used as the activity unit (e.g. Figure 5 ).

[0214] As shown in Figure (5), after the DNA sequence is connected to the magnetic beads and then amplified, the magnetic beads are washed after the amplification reaction and compared with the supernatant for IVTT reaction. The washed magnetic beads have almost no activity, while the activity of the supernatant is relatively high, indicating that after the DNA primer sequence is linked to the magnetic beads and then amplified, the activity of the DNA sequence linked to the magnetic beads is extremely low. This proves that the DNA magnetic beads prepared by the method of the present application have high activity.

[0215] Example 8: Microsphere Modification

[0216] Weigh 1-50 g of glass microspheres, ultrasonically clean them with distilled water for 1-3 times, add 5-250 mL of 1-2 mol / L NaOH solution to the cleaned glass microspheres, react at 70° C. for 1-2 h, wash them with distilled water for 5-10 times, add 5-200 mL of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) ethanol solution (5%-20%, v / v) to the cleaned glass microspheres, react for 12-72 hours, and then wash the glass microspheres with anhydrous ethanol and distilled water for 3-5 times to obtain modified glass microspheres A.

[0217] Example 9: Preparation of DNA samples

[0218] (1) Polyacrylic acid solution a configuration

[0219] a. Take 4 ml of polyacrylic acid solution (molecular weight 3000, mass fraction 50%), add ddH2O to 40 mL, add 1 mol / L NaOH to adjust the pH to 7.5, and then add pure water to 50 mL;

[0220] b. Take 230 μL of the solution into a test tube, add ddH2O to 10 mL, and shake thoroughly.

[0221] (2) Add 318 μL of polyacrylic acid solution to the DNA primer sequence (the experiment used artificially synthesized DNA, the specific sequence is GGCGTAGAGGAT, and the amino group was modified at the C6 position of the 5' end during synthesis), shake it in a vortex machine for 5 minutes, and let it stand for 25 minutes.

[0222] (3) Use the modified DNA primer sequence amplification (Ampi) to obtain the green fluorescent protein DNA sample.

[0223] Example 10: Preparation of DNA glass microspheres.

[0224] (1) MES-KOH buffer preparation

[0225] ①Put 1.952g of MES in a beaker;

[0226] ② Add about 60 mL of ddH2O into the beaker and stir to dissolve MES.

[0227] ③ Add 1 mol / L KOH solution into the beaker and adjust the pH to 5.6.

[0228] ④ Transfer the liquid in the beaker to a 100mL volumetric flask and make up to volume.

[0229] (2) Wash glass microspheres A (320 mesh, 45 μm) to reduce the pH to 7-8.

[0230] (3) Take 100 μl of the washed glass microspheres A (without liquid) into centrifuge tube a, wash them three times with MES-KOH buffer, and then centrifuge to remove the supernatant.

[0231] (4) Add 100 μl of MES-KOH buffer into centrifuge tube a.

[0232] (5) Take 200 μl of green fluorescent protein DNA sample and transfer it to another centrifuge tube b.

[0233] (6) Prepare activation solution: weigh 0.300 g of EDC and 0.150 g of NHS into a 15 mL centrifuge tube, add MES-KOH buffer to 10 mL, and shake thoroughly until the drugs are completely dissolved.

[0234] (7) Transfer 100 μl of activation solution to centrifuge tube b, shake on a vortex machine for 1 min, and let stand for 15 min.

[0235] (8) Transfer all the mixed liquid in centrifuge tube b to centrifuge tube a and shake centrifuge tube a manually to fully mix the contents.

[0236] (9) Place centrifuge tube a on a rotator and react for 24 hours at room temperature (preferably 15°C).

[0237] Example 11: Activity Verification

[0238] (1) Take out centrifuge tube a and place it on a test tube rack to wait for the microspheres to separate from the liquid.

[0239] (2) Transfer the supernatant to centrifuge tube c.

[0240] (3) Add ddH2O to centrifuge tube a and wash three times, then aspirate the supernatant.

[0241] (4) Centrifuge tube a and centrifuge tube c were sent for inspection, and the target protein was synthesized in the IVTT system (same as in Example 4).

[0242] Depend on Figure 6 It can be seen that three groups of parallel experiments were conducted on the 320-mesh microspheres, and the activities of the three groups of DNA microspheres in groups 1, 2, and 3 were respectively RFU3699, RFU4532, and RFU3893. The highest activity was in group 2, which was 106% of the PC activity. Even the lowest activity was in group 1, which reached 86.5% of the PC activity. It can be seen that the DNA glass microspheres of the present invention also have good reaction activity as DNA templates, and have good repeatability and stability.

[0243] Example 12: Verify the effect of ultrasonic vibration on the activity of DNA microspheres.

[0244] The other steps are similar to those in Example 10, except that step (4-1) is added between steps (4) and (5) in Example 10: the glass microspheres resuspended in the buffer solution after washing are placed in an ultrasonic oscillation device for 10 minutes. The other steps refer to Example 10. Afterwards, the activity of the DNA microspheres was verified in the manner of Example 11, and the effect of ultrasonic oscillation on the activity of DNA microspheres with different particle sizes was verified. Multiple parallel experiments were used for effective verification (see Figure 7 ).

[0245] Depend on Figure 7It can be seen that the activity of DNA microspheres linked after ultrasonic treatment, whether 320 mesh or 2000 mesh (7.5μm), is higher than that of DNA microspheres without ultrasonic treatment, and this conclusion still holds true after repeated experimental verification. The group with the largest improvement was 2000U2-1, with an RFU increase of 81.7%, and the lowest was 2000U2-2, with an RFU increase of 2.3%. The above experiments prove that ultrasonic vibration of the glass microsphere suspension before linking the glass microspheres with DNA can significantly improve the reaction activity of the DNA microspheres, and is applicable to microspheres of different particle sizes, while still maintaining good repeatability and stability.

[0246] Based on the above ideal embodiments of this application, the relevant staff can make various changes and modifications without departing from the technical concept of this application through the above description. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A DNA microsphere, characterized in that: include: Microsphere body, polymer connected to the surface of the microsphere body, and DNA bound to the polymer.

2. The DNA microsphere according to claim 1, characterized in that: The polymer binds to DNA via a functional group; the functional group is selected from carboxyl, hydroxyl, amino, sulfhydryl, a salt form of a carboxyl, a salt form of an amino, an ester group, or a combination of the aforementioned functional groups.

3. The DNA microsphere according to claim 1 or 2, characterized in that: The DNA is modified with the functional group.

4. The DNA microsphere according to any one of claims 1 to 3, characterized in that: The polymer is directly or indirectly connected to the outer surface of the microsphere body through a connecting group.

5. The DNA microsphere according to claim 4, characterized in that: The linking group is selected from one or a combination of substituted or unsubstituted siloxy groups, amide groups, ester groups, carbonyl groups, alkenyl groups, alkynyl groups, alkylene groups, ether groups, and thioether groups.

6. The DNA microsphere according to any one of claims 1 to 5, characterized in that: The surface of the microsphere body contains silicon or silicon oxide; preferably, the material of the microsphere body is selected from glass, ceramic or magnetic material. When the microsphere body is a magnetic microsphere, its surface is coated with silicon dioxide.

7. The DNA microsphere according to any one of claims 1 to 6, characterized in that: The size of the microsphere body is selected from any one of the following particle size scales or the range between any two particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm , 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm; the diameter size is the average value; In one preferred embodiment, the diameter of the microsphere body is selected from 0.1 to 10 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.2 to 6 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.4 to 5 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.5 to 3 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.2 to 1 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.5 to 1 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 1 μm to 1 mm; In one preferred embodiment, the average diameter of the microsphere body is 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, with a deviation of ±20%, more preferably ±10%.

8. The DNA microsphere according to any one of claims 1 to 7, characterized in that: The polymer is one or more of polyolefin, PEG, polyacrylic acid, polyamide or polyester.

9. The DNA microsphere according to any one of claims 1 to 8, characterized in that: The average molecular weight MW of the polymer is 1000-5000000; more preferably, the MW is 1000-100000; further preferably, the MW is 2000-50000; further preferably, the MW is 3000-10000.

10. The DNA microsphere according to any one of claims 1 to 9, characterized in that: The DNA is obtained by amplifying a DNA primer sequence. Preferably, the length of the DNA primer sequence is 6 to 30 bp; preferably, 9 to 21 bp. Furthermore, preferably, the DNA primer sequence is combined with the polymer through the functional group before the amplification is performed. Furthermore, the DNA primer sequence is combined with the polymer through the functional group for the amplification and then connected to the surface of the microsphere body to obtain the DNA microsphere.

11. The DNA microsphere according to claim 10, characterized in that: in, The DNA primer sequence is modified with the functional group; further, the functional group is an amino group, and the 5' end of the DNA primer sequence contains an amino group; preferably, it contains an amino group at the C2-C20 position of the 5' end; more preferably, it contains an amino group at the C5-C10 position of the 5' end; most preferably, it contains an amino group at the C6 position of the 5' end.

12. A method for preparing the DNA microsphere according to any one of claims 1 to 11, characterized in that: The method comprises the following steps: (1) chemically modifying the microsphere body by using an amino silane coupling agent to introduce an amino group into the outer surface of the microsphere body to form an amino-modified microsphere A; (2) modifying a DNA primer sequence to obtain a modified DNA primer sequence; (3) combining the modified DNA primer sequence with a polymer, and then amplifying the DNA to obtain a polymer-modified DNA; and (4) reacting the polymer-modified DNA with the amino-modified microsphere A to obtain the DNA microsphere.

13. The method for preparing DNA microspheres according to claim 13, characterized in that: In step (2), the 5' end of the DNA primer sequence is modified with the amino group; preferably, the amino group is modified at the C2-C20 position of the 5' end; more preferably, the amino group is modified at the C5-C10 position of the 5' end; most preferably, the amino group is modified at the C6 position of the 5' end.

14. The method for preparing DNA microspheres according to claim 12 or 13, wherein the length of the DNA primer sequence is 6 to 30 bp; preferably 9 to 21 bp, and further, the DNA primer sequence is GGCGTAGAGGAT.

15. The method for preparing DNA microspheres according to any one of claims 12 to 14, characterized in that: The polymer is a polyolefin polymer; preferably, the polyolefin polymer is selected from one or more of substituted or unsubstituted polypropylene, polyacrylic acid, polyacrylic acid salt, polyacrylic acid ester or polyacrylamide.

16. The method for preparing DNA microspheres according to any one of claims 12 to 15, characterized in that: In step (4), before reacting the polymer-modified DNA with microsphere A, the system containing microsphere A is first subjected to ultrasonic vibration. 17 . The method for preparing DNA microspheres according to claim 16 , wherein the ultrasonic oscillation time is 1 to 30 min, preferably 5 to 20 min; more preferably 5 to 10 min.

18. Use of the DNA microsphere according to any one of claims 1 to 11, wherein the DNA microsphere is used as a DNA template.

19. Use of the DNA microsphere according to any one of claims 1 to 11 as a DNA template in RNA transcription or protein synthesis.

20. The use according to claim 20, wherein the RNA transcription or protein synthesis is in vitro RNA or in vitro protein synthesis.

21. A cell-free in vitro synthesis system, comprising: (1) Cell extracts; (2) The DNA microsphere according to any one of claims 1 to 11.

22. A cell-free in vitro synthesis method, comprising preparing the system according to claim 21.

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