WO2019033946A1 - 过滤出口注射器内分散多孔颗粒与液体的相互作用和分离 - Google Patents

过滤出口注射器内分散多孔颗粒与液体的相互作用和分离 Download PDF

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WO2019033946A1
WO2019033946A1 PCT/CN2018/098936 CN2018098936W WO2019033946A1 WO 2019033946 A1 WO2019033946 A1 WO 2019033946A1 CN 2018098936 W CN2018098936 W CN 2018098936W WO 2019033946 A1 WO2019033946 A1 WO 2019033946A1
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liquid
porous particles
reaction
core rod
dispersed porous
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PCT/CN2018/098936
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English (en)
French (fr)
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黄志坚
吕常庆
罗旻
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杭州立昂科技有限公司
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D36/00—Filter circuits or combinations of filters with other separating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning

Definitions

  • the present invention relates to a method for interacting and separating dispersed porous particles with a liquid batch implemented in a syringe with a filtered outlet, particularly suitable for affinity purification preparation of biological macromolecules such as proteins and nucleic acids.
  • the solid phase medium can adopt a porous particle structure having a high specific surface area.
  • porous particulate materials are agarose, cellulose and silica.
  • Column chromatography based on stacking medium is a classical purification system, but it has the disadvantages of low efficiency and difficulty in controlling the interaction time and degree of solid phase medium and liquid interaction, resulting in slow process and easy introduction of chromatography column.
  • a series of problems such as blockage of bubbles or impurities or stickiness, large amount of media and high cost, and dilution of the final purified product.
  • Purification technology based on magnetic solid phase media can achieve efficient, non-clogging and controlled batch interaction and separation of dispersed particles and liquids, overcoming the shortcomings of the above-mentioned stacked medium chromatography column, but because of the effective range of common magnet attraction The limited, reliable sample volume is actually only a few milliliters.
  • syringes with filtered outlets can efficiently and conveniently implement and control batch interaction and separation of dispersed solid phase media with liquids (Analyst, 2014, 139, 6266).
  • the method realizes solid phase extraction, and the purpose thereof is to qualitatively or quantitatively detect the target molecule, and is not purified by the basic requirement of yield, so it adopts a solid solid phase medium with low binding efficiency, and is not noticed in the preparation demand.
  • Porous media reduces the amount of media used in a limited syringe space and increases the advantages and importance of preparing a sample volume or total.
  • the invention provides a simple, efficient and controllable method for batch interaction and separation of dispersed porous particles and liquid, and uses the reaction unit to realize the affinity purification preparation of the liquid sample molecules.
  • the reaction unit is based on a syringe with a filter outlet, the syringe is filled with dispersed porous particles, and the liquid sample is sucked and pushed out from the filter outlet by the back and forth movement of the core rod, and the dispersed porous particles and the sample are conveniently, efficiently and controllably respectively. Batch interaction and separation of liquids.
  • the reaction unit can constitute a reaction, such as binding, blocking, washing and elution between the dispersed porous particles and the molecule of the sample, according to its properties and effects, and the use of these reaction units alone or in combination can be particularly convenient, efficient and controllable. Affinity purification of the target molecule of the liquid sample is achieved.
  • the invention overcomes the disadvantages of inefficient, clogging and uncontrollable solid and liquid interaction and purification of the classical bulk dielectric column chromatography method, and also avoids the small volume interaction of the dispersed magnetic particles and the purification method. problem.
  • the invention is particularly suitable for affinity purification of biological macromolecules such as proteins and nucleic acids.
  • Corresponding preferred dispersed porous particulate materials include, but are not limited to, agarose, cellulose, and silica.
  • the ligand molecule is solidified on the dispersed porous particles by covalent or non-covalent means to obtain a solid phase medium which can be used for affinity purification; as a preferred affinity purification system, dispersion porous
  • the particles are combined with the impurity molecules in the sample, and the purified molecular liquid of interest can be collected and separated by the core rod.
  • the dispersed porous particles are combined with the target molecules in the sample and separated from the corresponding product liquid. Thereafter, the washing and elution reaction between the dispersed porous particles and the target molecule is performed, and finally the liquid discharged from the core rod is the purified target molecule.
  • the invention also provides a kit for interacting and separating dispersed porous particles and liquid sample batches which are particularly suitable for affinity purification of biological macromolecular samples such as proteins and nucleic acids.
  • the kit consists of part or all of the following members: syringe with filter outlet, dispersed porous particles, binding solution, washing solution, blocking solution, eluent.
  • Figure 1 An injector system with a membrane outlet for biomacrometic affinity purification, containing dispersed porous particles intercepted by a filter, the core rod moving back and forth to absorb liquid to achieve it and disperse porous particles solid phase medium A batch interaction occurs, or the liquid is drained to achieve its batch separation from the solid phase medium, realizing a solid-liquid interaction and separation of the reaction unit.
  • the purification of the target molecule is separated from the multi-molecular mixture.
  • the monoclonal antibody is extracted and purified from the hybridoma cell culture medium containing various hybrid proteins; the purification in a broad sense is to take the target molecule from a state. Transfer to another target state, such as the solidification of the Protein G protein molecule in the liquid phase by covalent coupling to the agarose particles, is a purification of molecular aggregation state changes.
  • the present invention uses a syringe 2 device comprising a filter membrane 4 outlet and dispersed porous particles 3 as shown in Figure 1 to effect a solid reaction interaction and separation.
  • the core rod 1 is twitched, and a sample of the liquid 5 is taken in from the outlet of the membrane 4, and the sample molecules are incubated with the dispersed porous particles 3 to undergo specific interaction, reaction or combination;
  • the reacted liquid 5 is pushed out from the outlet of the filter membrane 4 using the core rod 1, and the dispersed porous particles 3 after the reaction remain in the syringe 2.
  • the above reaction unit of the syringe 2 with the dispersed porous particles 3 and the outlet of the membrane 4 can be further classified according to the nature and effect of the reaction, such as but not limited to the following table.
  • reaction units may be used singly or in any combination, so that the present invention is particularly suitable for affinity purification of biological macromolecules such as proteins and nucleic acids, and specific types such as, but not limited to, the following table
  • the material of the dispersed porous particle 3 of the syringe 2 and the liquid 5 interaction and separation system of the present invention should be inert, that is, it should not undergo significant physical interaction or chemical reaction with the liquid sample molecule to avoid the target molecule being solid. Adsorption or denaturation of the phase surface results in a decrease in activity and yield, and ensures efficient separation of the impurity molecules from the molecule of interest.
  • the syringe 2 and the core rod 1 of the present invention should have sufficient mechanical strength to withstand the twitching friction and pressure and to maintain the shape and the sealing of the contact surface.
  • Common examples are plastic, glass or metal.
  • the preferred diameter of the syringe 2 is between 2 mm and 100 mm, preferably containing between 0.5 ml and 1000 ml of liquid volume.
  • the membrane 4 of the syringe 2 should have sufficient mechanical strength to withstand the pressure of the liquid flow. It is preferable that the membrane 4 has a pore diameter of 10 ⁇ m or more to ensure smooth filtration.
  • the filter membrane 4 prepared by a commonly used silicon-based or polymer material can be obtained from the market. Obtained (such as the sieve board manufactured by Shenzhen Comic Biotechnology Co., Ltd.).
  • the present invention is particularly concerned with the affinity purification of biological macromolecules such as proteins and nucleic acids, and the use of dispersed porous particle 3 media to achieve efficient interaction and binding of large surfaces inside and outside the particle with the molecule of interest.
  • the dispersed porous particles 3 have a particle diameter of between 10 and 100 ⁇ m and can be effectively trapped in the syringe 2 by the above-mentioned filter membrane 4; the pore size of the porous structure is sufficiently large to enable a preferred molecular weight of 1 kilodalton and 1 Biomolecules such as peptides, proteins, endotoxins, viruses, plasmids, and nucleic acids are in and out of thousands of Daltons; they must also have sufficient resistance to deformation and deformation, and are not significantly deformed under the liquid pressure of various syringe operations.
  • dispersed porous particle 3 affinity medium satisfying the above requirements, such as but not limited to agarose, cellulose, and silica, are commercially available from GE, Thermo Fisher, Qiagen, and the like.
  • biomacromolecular affinity purification systems of the present invention include, but are not limited to, the following table
  • the substantive content of the invention includes, but is not limited to, the following embodiments.
  • Example 1 Filtered outlet syringe 2
  • a disposable syringe 2 containing a liquid volume of 20 ml is readily available or custom-made on the market, and the syringe and the stem 1 are made of polypropylene which does not adsorb biomacromolecules such as proteins and nucleic acids.
  • the 20 micron pore size filter membrane 4 placed at the outlet of the syringe 2 is commercially available from Shenzhen Comma Biotechnology Co., Ltd., and is made of hydrophilized polyethylene which does not adsorb biomacromolecules such as proteins and nucleic acids. This syringe 2 was used in the following examples.
  • Pre-cleaning reaction before medium coupling Remove the plunger 1 of the syringe, put 4 ml of N-hydroxysuccinimide to activate Sepharose 4Fast Flow dispersed porous particles 3 medium wet glue (GE), put back the core rod 1, push the extrusion medium Storage liquid liquid 5; use the core rod 1 to draw 12 ml of cold 1 millimolar hydrochloric acid cleaning liquid 5, mix well with the medium and slosh for 10 minutes at room temperature, push the core rod 1 to squeeze the cleaning liquid 5; The above hydrochloric acid washing liquid 5 washing operation was repeated once.
  • GE medium wet glue
  • Media blocking reaction twitch the mandrel 1 to inhale 12 ml of 0.5 molar ethanolamine and 0.5 molar sodium chloride solution pH 8.3 liquid 5, mix well with the medium and sway for 2 hours at room temperature, push the core rod 1 out Liquid 5; twitching mandrel 1 inhaled 12 ml of 0.1 molar concentration of tromethamine liquid 5 buffer pH 8.5, fully mixed with the medium and incubated at room temperature for 10 minutes, push the core rod 1 to extrude the liquid 5; twitch core The rod is inhaled into 12 ml of 0.1 molar acetate buffer pH 4.0 liquid 5 (containing 0.5 molar sodium chloride), thoroughly mixed with the medium and incubated at room temperature for 10 minutes, pushing the core rod 1 to extrude the liquid 5; repeat The above-mentioned tromethamine buffer and the acetate buffer liquid 5 were washed twice.
  • Media cleaning reaction twitch the core rod 1 to inhale 12 ml of phosphate buffered saline solution pH 7.4 liquid 5, mix well with the medium and sway for 10 minutes at room temperature, push the core rod 1 to extrude the liquid 5; repeat the above cleaning reaction 2 Times.
  • Medium pretreatment cleaning reaction take out the syringe core rod 1, put 4 ml of poly- ⁇ -lysine cellulose dispersed porous particles 3 medium suspension (ThermoFisher), put back the core rod 1, push the extrusion medium storage liquid liquid 5; Using the core rod 1 to draw 12 ml of 0.2 molar sodium hydroxide and 95% ethanol solution liquid 5, mix well with the medium and sway for 2 hours at room temperature, push the core rod 1 to squeeze the solution liquid 5; twitch the core rod 1 Inhale 12 ml of 2 molar sodium chloride liquid 5, mix well with the medium and incubate at room temperature for 10 minutes, push the core rod 1 to squeeze out the solution liquid 5; twitch the core rod 1 to inhale 12 ml of endotoxin-free water, Mix well with the medium and incubate at room temperature for 10 minutes, push the core rod 1 to extrude the liquid 5; twitch the core rod 1 to inhale 12 ml of phosphat
  • Media binding reaction twitch the mandrel 1 to inhale 12 ml of 1 mg concentration of bovine immunoglobulin liquid 5 per ml (dissolved in phosphate buffered saline pH 7.4), mix well with the medium and incubate at room temperature for 1 hour, push The core rod 1 extrudes the solution liquid 5.
  • the liquid 5 extruded from the last syringe plunger 1 is the purified bovine immunoglobulin for endotoxin removal.
  • Medium pre-cleaning reaction push the core rod 1, extrude the liquid 5 of the medium suspension of the Protein G-separated agarose-dispersed porous particles 3 prepared in Example 2; twitch the core rod 1 to inhale 12 ml of phosphate buffered saline solution pH 7. 4 liquid 5. Mix well with the medium and incubate at room temperature for 10 minutes, push the core rod 1 to extrude the solution liquid 5; repeat the phosphate buffer solution liquid 5 washing step twice.
  • Media cleaning reaction twitch the core rod 1 to inhale 12 ml of phosphate buffered saline solution 5, mix well with the medium and sway for 10 minutes at room temperature, push the core rod 1 to extrude the liquid 5; repeat the phosphate buffer solution liquid 5 clean Step 2 times.
  • Media elution reaction twitch the mandrel 1 to inhale 4 ml of 0.1 molar citric acid buffer solution pH 3.0 liquid 5, mix well with the medium and incubate at room temperature for 10 minutes, push the core rod 1 to extrude the eluent liquid 5; If necessary, repeating the above elution reaction 1 to several times can increase the recovery of the mouse monoclonal antibody molecule remaining inside the pores of the dispersed porous particles 3 and between the particles and inside the syringe 2.
  • Example 5 Histidine tag recombinant protein is being purified
  • Medium pre-cleaning reaction take out the plunger 1 of the syringe, put in 4 ml of Ni Sepharose 6Fast Flow agarose-dispersed porous particles 3 medium suspension (GE), put back the core rod 1, push the extruded medium storage liquid liquid 5; use the core rod 1 Extract 12 ml of purified water liquid 5, mix well with the medium and incubate at room temperature for 10 minutes, push the core rod 1 to extrude the liquid 5; twitch the core rod 1 to inhale 12 ml of 20 millimolar phosphate buffer pH7. 4 Liquid 5 (containing 0.5 molar sodium chloride and 20 millimolar concentration of imidazole), thoroughly mixed with the medium and incubated at room temperature for 10 minutes, pushing the core rod 1 to extrude the liquid 5.
  • GE Ni Sepharose 6Fast Flow agarose-dispersed porous particles 3 medium suspension
  • Media binding reaction twitching mandrel 1 inhaled 12 ml of prepared histidine-tagged recombinant protein clear liquid 5 (dissolved in 20 mM phosphate buffer pH 7.4, containing 0.5 molar sodium chloride and 20 mM Imidazole), mixed well with the medium and incubated at room temperature for 1 hour, pushing the mandrel 1 to extrude the liquid 5.
  • Media cleaning reaction twitch the mandrel 1 to inhale 12 ml of 20 millimolar phosphate buffer pH 7.4 liquid 5 (containing 0.5 molar sodium chloride and 0.1 molar imidazole), mix well with the medium and incubate at room temperature 10 In minutes, the mandrel 1 was pushed to extrude the liquid 5; the washing reaction was repeated twice.
  • Media elution reaction twitch the mandrel 1 to inhale 4 ml of 20 millimolar phosphate buffer pH 7.4 liquid 5 (containing 0.5 molar sodium chloride and 0.4 molar imidazole), mix well with the medium and incubate at room temperature 10 minutes, pushing the core rod 1 to extrude the liquid 5; if necessary, repeating the above elution reaction 1 to several times, the histidine tag recombination remaining inside the pores of the dispersed porous particles 3 and between the particles and inside the syringe 2 can be increased. Recovery of protein molecules.
  • the eluent liquid 5 extruded from the core rod 1 is collected and collected, and the histidine-tagged recombinant protein is purified by dialysis or chromatographic desalting.
  • Hydrochloric acid buffer pH 8.0 (containing 10 millimolar concentration of ethylenediaminetetraacetic acid), repeatedly mixed with a pipette; add 300 ⁇ l of 1% sodium lauryl sulfate and 0.2 molar sodium hydroxide, gently reversed Mix well, let stand for 3 minutes at room temperature; add 300 ⁇ l of pre-cooled 2.5 molar guanidine hydrochloride and 0.75 molar potassium acetate pH 3.8, mix quickly by inversion, ice bath for 5 minutes, re-mix; 12,000g at 4 degrees Centrifuge for 10 minutes and take the supernatant for use.
  • Medium pre-cleaning reaction take out the plunger 1 of the syringe, put 1 gram of diethylaminoethyl-modified silica dispersed porous particles 3 anion exchange medium, put back the core rod 1, and draw 12 ml of Buffer QBT liquid with the core rod 1 The mixture was thoroughly mixed with the medium and incubated at room temperature for 10 minutes, and the core rod 1 was pushed out to extrude the solution liquid 5.
  • the wicking mandrel 1 was inhaled into the prepared plasmid crude liquid 5, mixed well with the medium and incubated at room temperature for 30 minutes, and the mandrel 1 was pushed out to extrude the solution liquid 5.
  • Media cleaning reaction twitch the core rod 1 to draw 12 ml Buffer QC liquid 5, mix well with the medium and sway for 10 minutes at room temperature, push the core rod 1 to extrude the solution liquid 5; repeat the liquid 5 to clean the reaction twice.
  • Media elution reaction twitch the mandrel 1 to inhale 4 ml of Buffer QF liquid 5, mix well with the medium and incubate for 10 minutes at room temperature, push the mandrel 1 to extrude the eluate liquid containing the plasmid 5; if necessary, By repeating the above elution reaction 1 to several times, the recovery of the plasmid molecules remaining inside the pores of the dispersed porous particles 3 and between the particles and inside the syringe 2 can be improved.
  • Post-treatment reaction 0.7 times volume of isopropanol was added to the collected eluate samples, mixed by inversion, centrifuged at 15,000 g for 30 minutes, and the supernatant was carefully discarded; the precipitated plasmid was washed by adding 1 ml of 70% ethanol, and centrifuged at 15,000 g. 10 minutes, carefully discard the supernatant; repeat the ethanol washing operation; air-dry for 10 minutes at room temperature, add 10 millimolar concentration of tromethamine buffer solution prepared without endotoxin water, pH 8.0 (containing 1 millimolar concentration of ethylenediamine) Tetraacetic acid) dissolved.
  • the mandrel 1 pushes the extrusion cleaning liquid 5 .
  • the core rod 1 is taken out, the outlet of the syringe syringe filter 4 is erected downward; 12 ml of the last used washing liquid is injected, and the suspended dispersed porous particles 3 are appropriately agitated in the downward flow of the liquid, taking care to remove the bubbles;
  • the outlet of the membrane 4 is closed; the eluent of the medium wet glue volume is added 1 to 3 times, the outlet of the membrane 4 is opened, and all the eluate samples are collected.
  • kits suitable for affinity purification of biological macromolecular samples such as proteins and nucleic acids are designed.
  • the kits each contain a syringe 2 with a filter membrane 4 outlet and a mandrel 1 and a dispersion of porous particles 3 medium, and the user provides a sample of liquid 5 to be purified.
  • the specific application kits are as follows.
  • (1) Medium coupling kit Covalent or non-covalent binding of the ligand sample liquid 5 to be coupled to the dispersed porous particles 3 having a functional reactive group to obtain a new dispersed porous particle having a ligand. Specific examples are as for example but not limited to embodiment 2.
  • Negative purification kit The impurities in the sample liquid 5 to be purified are combined with the dispersed porous particles 3 to obtain a purified sample liquid 5 discharged from the core rod 1, and the impurities are left in the syringe 2 together with the dispersed porous particles 3.
  • Specific examples are as for example but not limited to embodiment 3.

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Abstract

一种在带滤膜(4)出口以及分散多孔颗粒(3)的注射器(2)里实施的固体和液体相互作用和分离的方法，通过注射器(2)的芯杆(1)从滤膜(4)出口抽动吸入和推动排出液体(5)，实施了分散多孔颗粒(3)和液体(5)的相互作用和分离，可以实现清洗、结合、封闭和洗脱等反应，这些反应的组合构成适用于各类分子尤其是蛋白质和核酸等生物大分子的亲和纯化方法。以及一种含滤膜(4)出口和芯杆(1)的注射器(2)和分散多孔颗粒(3)的适用于样品亲和纯化的试剂盒。

Description

过滤出口注射器内分散多孔颗粒与液体的相互作用和分离 技术领域
本发明涉及一种在带有过滤出口的注射器里实现的分散多孔颗粒与液体批次相互作用和分离的方法，特别适合于生物大分子如蛋白质和核酸的亲和纯化制备。
背景技术
基于固相介质和液体相互作用和分离的分析和制备技术在分子科学中已经得到广泛的应用。生物大分子尤其是蛋白质不宜用温度和溶剂等物理方法进行纯化，因为它敏感的活性很容易被这些纯化手段所破坏。因此，在保持活性状态的要求下，液态生物大分子和它的固相化配体的亲和结合及分离成为常用的一种纯化它的方法。
为了提高固液相互作用的效率，固相介质可以采用高比表面积的多孔颗粒结构。对蛋白质和核酸一类的生物大分子纯化应用来说，最常用的多孔颗粒材料是琼脂糖、纤维素和二氧化硅。基于堆积介质的柱层析是经典的纯化体系，但它有固相介质和液体相互作用和分离的效率低并不易进行作用时间及程度控制的缺点，造成诸如过程缓慢、层析柱易被引入的气泡或杂物或粘性所堵塞、介质用量大而成本高以及最终纯化产品被稀释等一系列问题。基于磁性固相介质的纯化技术可以实现分散颗粒与液体的高效、无堵塞和可控的批次相互作用和分离，克服了上述堆积介质层析柱的缺点，但因为普通磁铁吸引力的有效范围有限，能可靠处理的样品体积实际上仅为几毫升。
我们注意到，带过滤出口的注射器可以高效方便地实现并控制分散固相介质与液体的批次相互作用和分离(Analyst,2014,139,6266)。该方法实现了固相萃取，其目的是对目的分子进行定性或定量检测，不是以产量为基本要求的纯化制备，因而它采用了低结合效率的实心固相介质，没有注意到在制备需求中多孔介质在有限注射器空间里降低介质使用量并提高可制备样品体积或总量的优势和重要性。事实上，即使固相萃取以检测为目的，使用分散的多孔颗粒作为与目的分子进行批次结合和分离的固相介质有大幅提高萃取效率的好处(Trends in Analytical Chemistry,2017,90,142)。
发明内容
本发明提供一种简便、高效和可控的分散多孔颗粒与液体的批次相互作用和分离的方法，并以此为反应单元实现液样目的分子的亲和纯化制备。该反应单元基于带过滤出口的注射器，其针筒内装有分散多孔颗粒，利用芯杆的来回运动从滤膜出口吸入和推出液体样品，方便、高效并可控地分别实现了分散多孔颗粒和样品液体的批次相互作用和分离。此反应单元根据其性质和效果可以构成针对分散多孔颗粒与样品目的分子之间的结合、封闭、清洗和洗脱等反应，而这些反应单元的单独或组合使用可特别方便、高效并可控地实现了液体样品目的分子的亲和纯化。本发明克服了经典堆积介质柱层析方法的固体和液体相互作用和纯化的低效、堵塞和不可控等缺点，也避免了分散磁性颗粒批次相互作用和纯化方法可处理样品体积偏小的问题。
本发明特别适用于蛋白质和核酸等目的生物大分子的亲和纯化。相应的优选分散多孔颗粒材料包括但不限于琼脂糖、纤维素和二氧化硅。作为优选亲和纯化体系之一，配体分子通过共价或非共价的方式固化在分散多孔颗粒上，得到可用于亲和纯化的固相介质；作为优选亲和纯化体系之二，分散多孔颗粒与样品中的杂质分子结合，被纯化的目的分子液体即可被芯杆分离排出而收集；作为优选亲和纯化体系之三，分散多孔颗粒与样品中目的分子结合并与相应的产物液体分离后，再执行分散多孔颗粒与目的分子之间的清洗和洗脱反应，最终由芯杆排出的液体即为纯化的目的分子。
本发明还提供一种特别适用于蛋白质和核酸等生物大分子样品亲和纯化的分散多孔颗粒与液样批次相互作用和分离的试剂盒。该试剂盒由下列组员的部分或全部构成：带滤膜出口的注射器、分散多孔颗粒、结合液、清洗液、封闭液、洗脱液。
附图说明
图1.特别适用于生物大分子亲和纯化的带有过滤膜出口的注射器系统，内含被滤膜拦截的分散多孔颗粒，其芯杆来回运动可以吸取液体实现它与分散多孔颗粒固相介质发生批次相互作用，或排出液体以实现它与该固相介质的批次分离，实现一个固液相互作用和分离的反应单元。
具体实施方式
狭义上的纯化是把目的分子从多分子的混合样中分离出来，比如单克隆抗体从含各种杂蛋白的杂交瘤细胞培养液中的提取纯化；广义上的纯化是把目的分子从一个状态转移到另一个目的状态，比如把游离于液相的Protein G蛋白分子通过共价偶联固化到琼脂糖颗粒上，就是一种分子聚集状态改变的纯化。
本发明使用图1所示的一种含过滤膜4出口和分散多孔颗粒3的注射器2装置来实现固液相互作用和分离这一基本反应单元。在第一步相互作用事件中，抽动芯杆1，从滤膜4出口吸入取液体5样品，让样品分子与分散多孔颗粒3温育并发生特定相互作用、反应或结合；在第二步分离事件中，使用芯杆1把反应后的液体5从滤膜4出口推出，而反应后的分散多孔颗粒3留在注射器2内。
上述的带分散多孔颗粒3和滤膜4出口的注射器2的反应单元可以根据反应的性质和效果进一步分类，如但不限于下表所示
Figure PCTCN2018098936-appb-000001
上述的反应单元可以单独或任何方式的组合使用，使得本发明特别适合蛋白质和核酸之类的生物大分子的亲和纯化，具体的类型如但不限于下表
Figure PCTCN2018098936-appb-000002
本发明所涉及的注射器2的分散多孔颗粒3与液体5相互作用和分离系统的材质应该具有惰性，即不应该和液体5样品分子发生显著的物理相互作用或化学反应，以避免目的分子在固相表面的吸附或变性而导致活性和得率的下降，并且保证杂质分子与目的分子的有效分离。
本发明所涉及的注射器2针筒和芯杆1的材质应该有足够的机械强度以承受抽动的摩擦和压力并保持形状以及接触面的密闭，常见的例子有塑料、玻璃或金属。注射器2的优选直径在2毫米和100毫米之间，优选容纳液体体积在0.5毫升到1000毫升之间。注射器2的滤膜4材质应有足够机械强度以承受液体流动的压力，优选滤膜4孔径在10微米以上以保证过滤的通畅，常用的硅基或高分子材料制备的滤膜4可以从市场上获得(如深圳逗点生物技术有限公司制造的筛板)。
本发明特别关注诸如蛋白质和核酸之类的生物大分子亲和纯化，采用分散多孔颗粒3介质来实现颗粒内外巨大表面与目的分子的高效率相互作用和结合。这种分散多孔颗粒3粒径在10至100微米之间，可被上述滤膜4有效截留在注射器2内；其多孔结构的孔径要足够大，能使优选分子量在1千道尔顿和1千万道尔顿之间的生物分子如多肽、蛋白质、内毒素、病毒、质粒和核酸等自由进出；它还必须具有足够的抗变形刚性，在各种注射器操作的液体压力下不显著变形，保持它对目的分子的结合力并减少颗粒之间因变形而产生的能截留液体的内闭空间；当然，这些介质还要能够被化学修饰而引入功能基团或配体。满足上述要求的分散多孔颗粒3亲和介质的例子如但不限于琼脂糖、纤维素和二氧化硅，均可从GE、ThermoFisher、Qiagen等处购得。
综上所述，本发明所涉及的优选生物大分子亲和纯化体系包括但不限于下表
Figure PCTCN2018098936-appb-000003
本发明的实质性内容包括但不限于以下实施例。
实施例1：带过滤出口注射器2
市场上容易购得或定制容纳液体体积为20毫升的一次性注射器2，其针筒和芯杆1材质均为不吸附蛋白质和核酸等生物大分子的聚丙烯。置于注射器2出口的20微米孔径的滤膜4可以从深圳逗点生物技术有限公司购得，其材质为不吸附蛋白质和核酸等生物大分子的经亲水化处理的聚乙烯。该注射器2用于以下实施例中。
实施例2：N-羟基琥珀酰亚胺活化琼脂糖与Protein G的介质偶联
介质偶联前预清洗反应：取出注射器芯杆1，放入4毫升N-羟基琥珀酰亚胺激活Sepharose 4Fast Flow分散多孔颗粒3介质湿胶(GE)，放回芯杆1，推挤出介质存储液液体5；用芯杆1抽取吸入12毫升冷1毫摩尔浓度盐酸清洗液液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出清洗液液体5；重复上述盐酸清洗液体5清洗操作1次。
介质偶联结合反应：抽动芯杆1吸入4毫升每毫升10毫克Protein G(Sigma)溶液液体5(溶于0.2摩尔浓度碳酸盐缓冲液pH8.3，含0.5摩尔浓度氯化钠)，与介质充分混匀并于室温下晃荡温育2小时，推动芯杆1挤出未反应的Protein G液体5。
介质封闭反应：抽动芯杆1吸入12毫升0.5摩尔浓度乙醇胺和0.5摩尔浓度氯化钠溶液pH8.3液体5，与介质充分混匀并于室温下晃荡温育2小时，推动芯杆1挤出液体5；抽动芯杆1吸入12毫升0.1摩尔浓度缓血酸胺液体5缓冲液pH8.5，与介质充分混匀并于室温下温育10分钟，推动芯杆1挤出液体5；抽动芯杆吸入12毫升0.1摩尔浓度醋酸缓冲液pH4.0液体5(含0.5摩尔浓度氯化钠)，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出液体5；重复上述缓血酸胺缓冲液和醋酸缓冲液液体5清洗反应2次。
介质清洗反应：抽动芯杆1吸入12毫升磷酸缓冲盐溶液pH7.4液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出液体5；重复上述清洗反应2次。
最终结果：抽动芯杆1吸入4毫升磷酸缓冲盐溶液pH7.4液体5(含20％乙醇防腐)，即得偶联有Protein G琼脂糖分散多孔颗粒3悬液，待用。
实施例3：牛免疫球蛋白样内毒素去除的负纯化
介质预处理清洗反应：取出注射器芯杆1，放入4毫升聚ε-赖氨酸纤维素分散多孔颗粒3介质悬液(ThermoFisher)，放回芯杆1，推挤出介质存储液液体5；用芯杆1抽取吸入12毫升0.2摩尔浓度氢氧化钠和95％乙醇溶液液体5，与介质充分混匀并于室温下晃荡温育2小时，推动芯杆1挤出溶液液体5； 抽动芯杆1吸入12毫升2摩尔浓度氯化钠液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出溶液液体5；抽动芯杆1吸入12毫升无内毒素水，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出液体5；抽动芯杆1吸入12毫升磷酸缓冲盐溶液pH7.4液体5，与介质充分混匀并于室温下温育10分钟，推动芯杆1挤出溶液液体5；重复磷酸缓冲盐溶液液体5清洗步骤2次。
介质结合反应：抽动芯杆1吸入12毫升每毫升1毫克浓度牛免疫球蛋白液体5(溶于磷酸缓冲盐溶液pH7.4)，与介质充分混匀并于室温下晃荡温育1小时，推动芯杆1挤出溶液液体5。
最终结果：最后注射器芯杆1挤出的液体5即为内毒素去除的纯化牛免疫球蛋白。
实施例4：小鼠单抗正纯化
介质预清洗反应：推动芯杆1，挤出实施例2所制备的偶联有Protein G的琼脂糖分散多孔颗粒3介质悬浮液之液体5；抽动芯杆1吸入12毫升磷酸缓冲盐溶液pH7.4液体5。与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出溶液液体5；重复磷酸缓冲盐溶液液体5清洗步骤2次。
介质结合反应：小鼠单抗腹水液2毫升于15,000g离心5分钟，取上清并加入2毫升磷酸缓冲盐溶液；抽动芯杆1吸入该单抗液样液体5，与介质充分混匀并于室温下晃荡温育1小时，推动芯杆1挤出溶液液体5。
介质清洗反应：抽动芯杆1吸入12毫升磷酸缓冲盐溶液液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出液体5；重复磷酸缓冲盐溶液液体5清洗步骤2次。
介质洗脱反应：抽动芯杆1吸入4毫升0.1摩尔浓度柠檬酸缓冲溶液pH3.0液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出洗脱液液体5；如有必要，重复上述洗脱反应1到数次，可以提高残留在分散多孔颗粒3孔径内部和颗粒之间以及注射器2内部的小鼠单抗分子的回收率。
最终结果：立即在汇总收集的洗脱液样加入1/10体积1摩尔浓度缓血酸胺缓冲液pH9.5实施中和反应，再经过透析或层析脱盐，即得纯化小鼠单抗样品。
实施例5：组氨酸标签重组蛋白正纯化
介质预清洗反应：取出注射器芯杆1，放入4毫升Ni Sepharose 6Fast Flow琼脂糖分散多孔颗粒3介质悬浮液(GE)，放回芯杆1，推挤出介质存储液液体5；用芯杆1抽取吸入12毫升纯净水液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出液体5；抽动芯杆1吸入12毫升20毫摩尔浓度磷酸缓冲液pH7.4液体5(含0.5摩尔浓度氯化钠和20毫摩尔浓度咪唑)，与介质充分混匀并于室温下温育10分钟，推动芯杆1挤出液体5。
介质结合反应：抽动芯杆1吸入12毫升制备好的组氨酸标签重组蛋白澄清液液体5(溶于20毫摩尔浓度磷酸缓冲液pH7.4，含0.5摩尔浓度氯化钠和20毫摩尔浓度咪唑)，与介质充分混匀并于室温下温育1小时，推动芯杆1挤出液体5。
介质清洗反应：抽动芯杆1吸入12毫升20毫摩尔浓度磷酸缓冲液pH7.4液体5(含0.5摩尔浓度氯化钠和0.1摩尔浓度咪唑)，与介质充分混匀并于室温下温育10分钟，推动芯杆1挤出液体5；重复该清洗反应2次。
介质洗脱反应：抽动芯杆1吸入4毫升20毫摩尔浓度磷酸缓冲液pH7.4液体5(含0.5摩尔浓度氯化钠和0.4摩尔浓度咪唑)，与介质充分混匀并于室温下温育10分钟，推动芯杆1挤出液体5；如有必要，重复上述洗脱反应1到数次，可以提高残留在分散多孔颗粒3孔径内部和颗粒之间以及注射器2内部的组氨酸标签重组蛋白分子的回收率。
最终结果：芯杆1挤出的洗脱液液体5汇总收集，经过透析或层析脱盐处理即为纯化组氨酸标签 重组蛋白。
实施例6：转染级质粒提取和正纯化
质粒粗样制备：用2毫升灭菌离心管收集1.5毫升含pUC18质粒的JM109菌株的过夜培养液，经过12000g离心1分钟，吸弃上清液；加入300微升50毫摩尔浓度缓血酸胺盐酸缓冲液pH8.0(含10毫摩尔浓度乙二胺四乙酸)，用移液枪反复吹打混匀；加入300微升1％十二烷基硫酸钠和0.2摩尔浓度氢氧化钠，温和颠倒混匀，室温静置裂解3分钟；加入300微升预冷2.5摩尔浓度盐酸胍和0.75摩尔浓度醋酸钾pH3.8，快速颠倒混匀，冰浴5分钟，重新混匀；4度下12,000g离心10分钟，取上清液备用。
介质与缓冲液：二乙氨乙基修饰的二氧化硅分散多孔颗粒3阴离子交换介质、平衡液Buffer QBT，清洗液Buffer QC，洗脱液QF均购自Qiagen。
介质预清洗反应：取出注射器芯杆1，放入1克二乙氨乙基修饰二氧化硅分散多孔颗粒3阴离子交换介质，放回芯杆1，用芯杆1抽取吸入12毫升Buffer QBT液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出溶液液体5。
介质结合反应：抽动芯杆1吸入上述制备好的质粒粗样液体5，与介质充分混匀并于室温下温育30分钟，推动芯杆1挤出溶液液体5。
介质清洗反应：抽动芯杆1吸取12毫升Buffer QC液体5，与介质充分混匀并于室温下晃荡温育10分钟，推动芯杆1挤出溶液液体5；重复该液体5清洗反应2次。
介质洗脱反应：抽动芯杆1吸入4毫升Buffer QF液体5，与介质充分混匀并于室温下温育10分钟，推动芯杆1挤出含质粒的洗脱液液体5；如有必要，重复上述洗脱反应1到数次，可以提高残留在分散多孔颗粒3孔径内部和颗粒之间以及注射器2内部的质粒分子的回收率。
后处理反应：在汇总收集的洗脱液样中加入0.7倍体积异丙醇，颠倒混匀，15,000g离心30分钟，小心吸弃上清；加入1毫升70％乙醇清洗沉淀质粒，15,000g离心10分钟，小心吸弃上清；重复乙醇清洗操作一次；室温晾干10分钟，加入无内毒素水配制的10毫摩尔浓度缓血酸胺缓冲液pH8.0(含1毫摩尔浓度乙二胺四乙酸)溶解。
最终结果：纯化质粒液样。
实施例7：纯化目的分子的高效率层析洗脱回收
在实施例4、5和6的正纯化中，注射器芯杆1推挤洗脱液液体5后，仍有部分含目标分子的洗脱液液体5残留在分散多孔颗粒之间和颗粒内部孔道的空间中。如果液体5目的分子的浓度或粘稠度比较高，这部分残留损失会比较大，用上述多次洗脱的方法则造成最终目的分子样品的稀释。如果基于这种注射器的洗脱反应改为下述的层析洗脱反应，分布在各空间的目的分子基本上可以全部被洗脱液定向置换推洗出来，目的分子的回收率高。因此，目的分子高回收率的纯化步骤修饰如下。
介质预清洗反应：如实施例4、5或6所述。
介质结合反应：如实施例4、5或6所述。
介质清洗反应：如实施例4、5或6所述。
介质层析洗脱反应：在介质清洗反应的最后一步，芯杆1推动挤出清洗液液体5。在完成这一步后，取出芯杆1，将注射器针筒滤膜4出口朝下直立；注入12毫升最后用的清洗液，在液体下降流动中适当搅动悬浮的分散多孔颗粒3，注意清除气泡；在清洗液液面下降到固相介质界面时关闭滤膜4出口；加入介质湿胶体积1到3倍的洗脱液，打开滤膜4出口，收集流出的全部洗脱液样。
洗脱液样后处理：如实施例4、5或6所述。
实施例8：纯化试剂盒
根据本发明的实质设计适用于蛋白质和核酸等生物大分子样品亲和纯化的试剂盒。该试剂盒均含有带滤膜4出口和芯杆1的注射器2和分散多孔颗粒3介质，用户提供待纯化的液体5样品。具体应用的试剂盒如下述。
(1)介质偶联试剂盒：待偶联的配体样品液体5与有功能活性基团的分散多孔颗粒3的共价或非共价结合，得到新的具有配体的分散多孔颗粒。具体例子如但不限于实施例2。
(2)负纯化试剂盒：待纯化的样品液体5中的杂质与分散多孔颗粒3结合，得到芯杆1排出的纯化样品液体5，杂质与分散多孔颗粒3一道留在注射器2内。具体例子如但不限于实施例3。
(3)正纯化试剂盒：待纯化的样品液体5中的目的分子与分散多孔颗粒3结合，再经过清洗和洗脱，最后得到芯杆1排出的纯化样品液体5。具体例子如但不限于实施例4、5和6。

Claims (25)

  1. 一种固体和液体相互作用和分离的方法，其特征为在一个带有分散多孔颗粒(3)固体介质和滤膜(4)出口的注射器(2)里，芯杆(1)抽动吸入液体(5)与分散多孔颗粒(3)发生相互作用，芯杆(1)推动排出作用后的液体(5)与分散多孔颗粒(3)发生分离。
  2. 根据权利要求1所述的方法，其特征为所述的相互作用和分离实现了分散多孔颗粒(3)在清洗液液体(5)中的清洗反应。
  3. 根据权利要求1所述的方法，其特征为所述的相互作用和分离实现了分散多孔颗粒(3)与样品液体(5)分子的共价或非共价的结合反应。
  4. 根据权利要求1所述的方法，其特征为所述的相互作用和分离实现了分散多孔颗粒(3)与封闭液液体(5)分子的共价或非共价封闭反应。
  5. 根据权利要求1所述的方法，其特征为所述的相互作用和分离实现了分散多孔颗粒(3)在洗脱液液体(5)中的洗脱反应。
  6. 一种固相介质偶联配体的方法，其特征为含有如下次序的操作步骤：
    (1)在一个带分散多孔颗粒(3)固相介质和滤膜(4)出口的注射器(2)里，抽动芯杆(1)吸入配体液体(5)并与分散多孔颗粒(3)发生共价或非共价偶联的结合反应，推动芯杆(1)排出反应后的配体液体(5)；
    (2)抽动芯杆(1)吸入封闭液液体(5)并与分散多孔颗粒(3)发生共价或非共价偶联的结合反应，推动芯杆(1)排出反应后的封闭液液体(5)；
    (3)抽动芯杆(1)吸入清洗液液体(5)并与分散多孔颗粒(3)发生清洗反应，推动芯杆(1)排出清洗液液体(5)，留在注射器(2)里的带配体的分散多孔颗粒(3)即为目的固相介质。
  7. 根据权利要求6所述的方法，其特征为所述的分散多孔颗粒(3)固相介质是琼脂糖、纤维素或二氧化硅。
  8. 根据权利要求7所述的方法，其特征为所述的琼脂糖带有能和带胺基配体共价介质偶联反应的N-羟基琥珀酰亚胺。
  9. 一种负纯化方法，其特征为在一个带有分散多孔颗粒(3)固相介质和滤膜(4)出口的注射器(2)里，抽动杆芯(1)吸入样品液体(5)并与分散多孔颗粒(3)发生共价或非共价结合反应，推动芯杆(1)排出反应后纯化的目的样品液体(5)。
  10. 根据权利要求9所述的方法，其特征为所述的分散多孔颗粒(3)固相介质是琼脂糖、纤维素或二氧化硅。
  11. 根据权利要求10所述的方法，其特征为所述的纤维素带有能结合并去除内毒素的聚ε赖氨酸。
  12. 一种正纯化方法，其特征为含有如下次序的操作步骤：
    (1)在一个带分散多孔颗粒(3)固相介质和滤膜(4)出口的注射器(2)里，抽动芯杆(1)吸入样品液体(5)并与分散多孔颗粒(3)发生结合反应，推动芯杆(1)排出反应后的样品液体(5)；
    (2)抽动芯杆(1)吸入清洗液液体(5)并与分散多孔颗粒(3)发生清洗反应，推动芯杆(1)排出反应后的清洗液液体(5)；
    (3)抽动芯杆(1)吸入洗脱液液体(5)并与分散多孔颗粒(3)发生洗脱反应，推动芯杆(1)排出纯化的目的洗脱液液体(5)。
  13. 根据权利要求12所述的方法，其特征为所述的分散多孔颗粒(3)固相介质是琼脂糖、纤维素或二 氧化硅。
  14. 根据权利要求13所述的方法，其特征为所述的琼脂糖带有能结合并纯化免疫球蛋白的Protein A、Protein G或Protein L，结合并纯化组氨酸标签重组蛋白的镍，或结合并纯化谷胱甘肽转移酶的谷胱甘肽。
  15. 根据权利要求13所述的方法，其特征为所述的二氧化硅带有能结合并纯化核酸的二乙氨乙基。
  16. 一种纯化方法，其特征为含有如下次序的操作步骤：
    (1)在一个带分散多孔颗粒(3)固相介质和滤膜(4)出口的注射器(2)里，抽动芯杆(1)吸入样品液体(5)并与分散多孔颗粒(3)发生结合反应，推动芯杆(1)排出反应后的配体液体(5)；
    (2)抽动芯杆(1)吸入清洗液液体(5)并与分散多孔颗粒(3)发生清洗反应，推动芯杆(1)排出反应后的清洗液液体(5)；
    (3)取出杆芯(1)，滤膜(4)出口朝下竖立注射器针筒，放入洗脱液层析洗脱分散多孔颗粒(3)，收集流出的纯化目的洗脱液。
  17. 根据权利要求16所述的方法，其特征为所述的分散多孔颗粒(3)固相介质是琼脂糖、纤维素或二氧化硅。
  18. 根据权利要求17所述的方法，其特征为所述的琼脂糖带有能结合并纯化免疫球蛋白的Protein A、Protein G或Protein L，结合并纯化组氨酸标签重组蛋白的镍，或结合并纯化谷胱甘肽转移酶的谷胱甘肽。
  19. 根据权利要求17所述的方法，其特征为所述的二氧化硅带有能结合并纯化核酸的二乙氨乙基。
  20. 一种纯化试剂盒，其特征为含有如下组分：
    (1)带滤膜(4)出口和芯杆(1)的注射器(2)；
    (2)分散多孔颗粒(3)固相介质。
  21. 根据权利要求20所述的试剂盒，其特征为所述的分散多孔颗粒(3)固相介质是琼脂糖、纤维素或二氧化硅。
  22. 根据权利要求21所述的试剂盒，其特征为所述的琼脂糖带有能结合并正纯化免疫球蛋白的Protein A、Protein G或Protein L，结合并正纯化组氨酸标签重组蛋白的镍，或结合并正纯化谷胱甘肽转移酶的谷胱甘肽。
  23. 根据权利要求21所述的试剂盒，其特征为所述的二氧化硅带有能结合并正纯化核酸的二乙氨乙基。
  24. 根据权利要求21所述的试剂盒，其特征为所述的纤维素带有能结合并负纯化去除内毒素的聚ε赖氨酸。
  25. 根据权利要求21所述的试剂盒，其特征为所述的琼脂糖带有能和带胺基配体共价介质偶联反应的N-羟基琥珀酰亚胺。
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