CN106732371A - The preparation method of DNA immunization adsorbent - Google Patents

The preparation method of DNA immunization adsorbent Download PDF

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Publication number
CN106732371A
CN106732371A CN201611202976.2A CN201611202976A CN106732371A CN 106732371 A CN106732371 A CN 106732371A CN 201611202976 A CN201611202976 A CN 201611202976A CN 106732371 A CN106732371 A CN 106732371A
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dna
resin
solution
preparation
nano
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CN106732371B (en
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董凡
谢高峰
黄杰辉
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Jafron Biomedical Co Ltd
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Jafron Biomedical Co Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes

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Abstract

The present invention provides a kind of preparation method of DNA immunization adsorbent, it is poured into DNA coating solution by by dry carbide resin, then stirring, filtering, drying add ethanol or ethanol ether mixed solvent, stirring, filtering, dry, cooling, obtain finished product DNA immunization adsorbent.The method uses " wet corrosion " coating method, after a coating is carried out to adsorbent, add appropriate organic solvent portion dissolving coating solution, so as to the DNA " local release " of film surface will be attached to, make the film forming again of the coating solution after dissolving, make membrane wrapping thickness more uniform, the effect of the effective adsorption rates of DNA is improved so as to reach.

Description

The preparation method of DNA immunization adsorbent
Technical field
The present invention relates to field of blood purification, and in particular to a kind of preparation method of carbide resin DNA immunity adsorbent.
Background technology
Systemic loupus erythematosus (systemiclupuserythematosus, hereinafter referred to as SLE) is a kind of common tired And the autoimmune disease of multisystem multiple organ.Because patient's Organism immunoregulation obstacle and autoimmune tolerance state are beaten It is broken, various antibody for autologous tissue are produced, cause the damage of tissue and organ, these antibody are mainly anti-Sm antibody, resist Antinuclear antibodies and ADA.Wherein ADA can be divided into anti-ssDNA antibody (anti-ss-DNA antibody) and anti-double-chain DNA Antibody (anti-ds-DNA antibody).
Shown according to domestic and international clinical and basic research, titre and disease severity and the activity of anti-ds-DNA antibody It is proportionate.The application of recent years, hormone and immunodepressant make SLE patient more after effect be improved significantly, but according to Old to there are many inapparent phenomenons of Patient drug's curative effect, the infection caused by excessive immunosuppressive therapy is to cause SLE to suffer from Person's main causes of death.In addition, many-sided research confirms that anti-dsDNA antibody (anti-ds-DNA antibody) is combined into DNA Immune complex is deposited in GBM, or ds-DNA antibody directly acts on glomerulus antigen, so as to cause SLE patient Injury of kidney.
Adsorb (IA) using DNA immunization carries out blood perfusion to patient, is the new treatment for growing up for nearly two, 30 years Method.Jones, Frank R (EP0272792A1,1987) are explained in detail to this method.The absorption of DNA immunization adsorbent Effect is the specific recognition effect to pathogenic anti-ds-DNA antibody by DNA, removes the morbid substance in blood samples of patients, is alleviated The state of an illness, improves the immunologic function of patient, so as to gradually recover one's health.
The TermanDS in the U.S. et al. (Lancet, 1979,2 (8147):It is carrier material 824-7) to use activated carbon first Material, using collodion coating fixed dna, obtains DNA immunization adsorbent for one severe SLE patient of hemoperfusion treatment, makes Patient vitals extend, and the beginning that DNA adsorbents treat SLE is opened from this.Using collodion coating, though adsorb DNA immunization The safety and effectiveness of agent is guaranteed, but adsorption rate is only 50% or so.
The content of the invention
The technical problem to be solved in the present invention is to provide and a kind of improves the new DNA immunization of DNA immunization adsorbent adsorption rate The preparation method of adsorbent.
The present invention provides a kind of preparation method of DNA immunization adsorbent, comprises the following steps：
Step one：Adsorbent coating
Be poured into dry carbide resin in DNA coating solution by step 1.1, stirring, filtering, drying, wherein the carbon It is 1 to change resin with the volume ratio of the DNA coatings solution：(2-4)；
Step 1.2 to adding organic solvent in step 1.1 products therefrom, the body of the carbide resin and the organic solvent Product is than being 1：(0.5-1), the organic solvent is ethanol or ethanol ether mixed solvent, second in the ethanol ether mixed solvent The concentration of volume percent of alcohol is at least 60%；
Step 2：By the stirring of step one products therefrom, filtering, dry, cooling, finished product DNA immunization adsorbent is obtained.
The above method uses " wet corrosion " coating method, after a coating is carried out to adsorbent, adds appropriate organic molten Agent is partly dissolved coating solution, so as to attach to the DNA " local release " of film surface, makes the coating solution after dissolving again Film forming, makes membrane wrapping thickness more uniform, fixed dna, and the effect of the effective adsorption rates of DNA is improved so as to reach.Wherein, organic solvent Can be ethanol or ethanol ether mixed solvent.When organic solvent is ethanol ether mixed solvent, ethanol is excellent in mixed solvent The concentration of volume percent of choosing is to be not less than 60%.It is more highly preferred to, organic solvent is ethanol ether mixed solvent, wherein ethanol Concentration of volume percent be 70%-90%, in such cases, adsorption rate is up to 90%.
More preferable scheme is that the method that the DNA coatings solution in step one passes through to comprise the steps of is prepared：
Step A is to the amino acid that the 0.1%-2% equivalent to DNA mass is added in the DNA stostes that concentration is 1-2mg/ml And the absolute ethyl alcohol isometric with DNA stostes, stirring or vibration obtain DNA solution to being completely dissolved；
Step B is by the collodion solution of step A gained DNA solution and 0.5%-1% mass concentrations (0.25- by volume 0.5)：1 is sufficiently mixed, and obtains DNA coating solution.
Good scheme is that amino acid described in step A is polar amino acid again, selected from glycine, serine, threonine, junket One or more in propylhomoserin, aspartic acid, cysteine.More preferably glycine.
Amino acid can improve dissolubilities of the DNA in water, therefore add a certain amount of amino acid, Ke Yigai in coating solution The solubility property of kind DNA, reaches the effect of homogeneous phase solution.Wherein, glycine is a kind of essential amino acid, substantially nontoxic pair Effect, meanwhile, glycine can also greatly improve the dielectric constant of the aqueous solution, so as to improve dissolubilities of the DNA in water.
Carbide resin in another preferable technical scheme steps one is obtained by polymerization, in polymerization, to polymerization system Middle addition nano-particle is used as template.
By will be added in the copolymerization system for preparing macroporous absorbent resin as the nano-particle of template, can make The polymeric adsorbent rich in nanometer mesopore prepared.After resin is carbonized in Muffle furnace, sour fully removal mould is carried out to resin Plate agent, you can prepare rich in the controllable carbide resin of nanometer mesopore pore size.
Another preferably technical scheme is that the method that the carbonized resin in step one passes through to comprise the steps of is obtained：
Step a prepares polystyrene resin
Initiator, nano template are added in the mixed system that Xiang Youshui phases and oil phase are constituted, be to slowly warm up to 95 DEG C- 100 DEG C are reacted 3-5 hour, through filtering, after carrying out washing treatment, preparing polystyrene resin；
Wherein, the water is mutually the aqueous gelatin solution that mass concentration is 0.5%-5%；The oil phase is styrene and diethyl The mix monomer of alkene benzene, the divinylbenzene accounts for the 0.5-1.5% of mix monomer gross mass；Water phase and oil phase volume ratio are (3- 4)：1；The consumption of the initiator is the 1-3% of mix monomer gross mass, and it is total that the consumption of the nano template accounts for mix monomer The 0.5-1% of quality；
The preparation of step b carbide resins
Polystyrene resin obtained in adding step a in resistance furnace, gradually heats up, with 80-100 DEG C under nitrogen protection Programming rate hourly is raised to 800-1000 DEG C, steam activation 1-2 hours；0.5- is boiled with 1-3mol/L HCl/water solution 1 hour, neutrality is washed to distillation, obtains carbide resin.
More preferred scheme is that nano-particle is nano-carbonate or Nanometer hydroxide.The nanoparticle being more highly preferred to Son can be Nano-meter CaCO33、MgCO3、BaCO3、SrCO3、ZnCO3、Mg(OH)2、Bi(OH)3、Fe(OH)3、Ti(OH)3、Cd (OH)2、Co(OH)2、Cr(OH)3、Mn(OH)2In any or it is various.It is still preferred that, the particle diameter of nano-particle is 2- 50nm。
The present invention does not have strict limitation to the initiator in the above method, and the initiator in wherein step a can be BP, the initiator in step b can be PBO.The present invention does not have to the compound method of the DNA stostes in the above method Strict border system, can use the method for preparing DNA stostes in the prior art, such as：By high-purity calf thymus DNA (DNA purity More than 85%) it is dissolved in 0.2mol/LTris-HCL (pH=7.4) cushioning liquid, compound concentration is that DNA concentration is 1-2mg/ Ml DAN stostes.
Temperature mentioned by above and below of the present invention, refers both to Celsius temperature.
Specific embodiment
With reference to embodiment, the present invention is further described.
Embodiment 1 prepares the DNA immunization adsorbent that sample number into spectrum is No. 1
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 2 times of carbide resin, stirring 5min and then filtering, dry 2 hours or so at the resin after filtering is placed on into 60 DEG C, and a DNA for coating is obtained after cooling Immunosorbent.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 2mg/ml.Isometric absolute ethyl alcohol is added toward the DNA stostes for preparing, is stirred Or vibration is to being completely dissolved, as DNA solution.
By DNA solution and 1% collodion solution by volume 0.25：1 is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 0.5% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 3：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 10nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour boils half an hour with the 3mol/LHCl aqueous solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Embodiment 2 prepares the DNA immunization adsorbent that sample number into spectrum is No. 2
The method that the present embodiment prepares No. 2 DNA immunization adsorbents is substantially the same manner as Example 1, and difference is in step The template agent that rapid a prepares used in polystyrene resin is the particle diameter about Nano-meter CaCO3 of 50nm3。
Embodiment 3 prepares the DNA immunization adsorbent that sample number into spectrum is No. 3
The method that the present embodiment prepares No. 3 DNA immunization adsorbents is substantially the same manner as Example 1, and difference is in step It is the nanometer Mg (OH) of particle diameter about 10nm or so that rapid a prepares the template agent used in polystyrene resin2。
Embodiment 4 prepares the DNA immunization adsorbent that sample number into spectrum is No. 4
The method that the present embodiment prepares No. 4 DNA immunization adsorbents is substantially the same manner as Example 1, and difference is in step It is the nanometer Mg (OH) of particle diameter about particle diameter about 50nm or so that rapid a prepares the template agent used in polystyrene resin2。
Embodiment 5 prepares the DNA immunization adsorbent that sample number into spectrum is No. 5
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 4 times of carbide resin, stirring 10min, then filters, and the resin after filtering is placed on cooling after being dried 2 hours at 60 DEG C a DNA immunization for coating is obtained Adsorbent.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 1mg/ml.Glycine is added toward the DNA stostes for preparing (equivalent to DNA stostes Middle DNA mass 0.1%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1% collodion solution by volume 0.25：1 is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 0.5% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 4：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 4 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 3% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour boils half an hour, with distillation with 1.5mol/L HCl/water solution Neutrality is washed to, carbide resin is obtained.
Embodiment 6 prepares the DNA immunization adsorbent that sample number into spectrum is No. 6
The method that the present embodiment prepares No. 6 DNA immunization adsorbents is substantially the same manner as Example 5, and difference is coating In the preparation process of liquid in the DNA stostes for preparing add be serine (equivalent to 0.1% of DNA mass in DNA stostes) And the absolute ethyl alcohol isometric with DNA stostes.
Embodiment 7 prepares the DNA immunization adsorbent that sample number into spectrum is No. 7
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 2 times of resin, stirs 1min mistakes The ethanol ether mixed solvent that resin volume ratio is 1/1 is added after filter, the concentration of volume percent of ethanol is 60%, will be filtered Cooling is DNA immunization adsorbent after resin afterwards is dried 2 hours at being placed on 60 DEG C.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 1mg/ml.Tyrosine is added toward the DNA stostes for preparing (equivalent to DNA stostes Middle DNA mass 0.1%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1.5% collodion solution by volume 0.5：1 (50%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 0.5% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 3：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 5 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour boils half an hour with 3mol/L HCl/water solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Embodiment 8 prepares the DNA immunization adsorbent that sample number into spectrum is No. 8
The method that the present embodiment prepares No. 8 DNA immunization adsorbents is substantially the same manner as Example 7, and difference is coating During in ethanol ether mixed solvent the concentration of volume percent of ethanol be 70%.
Embodiment 9 prepares the DNA immunization adsorbent that sample number into spectrum is No. 9
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 4 times of resin, stirs 5min, The ethanol ether mixed solvent that resin volume ratio is 1/1 is added after filtering, the concentration of volume percent of ethanol is 80%, stirring 1min, the resin after filtering is placed on cooling after being dried 2 hours at 60 DEG C and obtains the DNA immunization adsorbent that sample number into spectrum is No. 9.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 1mg/ml.Glycine is added toward the DNA stostes for preparing, and (consumption is DNA stostes Middle DNA mass 0.1%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1% collodion solution by volume 0.25：1 (25%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 0.5% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 3：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 100 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, The consumption of benzoyl peroxide is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 1000 DEG C, and steam activation 1 hour boils half an hour, with distillation with 3mol/L HCl/water solution Neutrality is washed to, carbide resin is obtained.
Embodiment 10 prepares the DNA immunization adsorbent that sample number into spectrum is No. 10
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 2 times of resin, stirs 10min, The ethanol ether mixed solvent that resin volume ratio is 1/1 is added after filtering, proportion of ethanol is 90%, 1min is stirred, after filtering Resin be placed at 60 DEG C dry 2 hours after cooling obtain the DNA immunization adsorbent that sample number into spectrum is No. 10.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 2mg/ml.Threonine is added toward the DNA stostes for preparing, and (consumption is DNA stostes Middle DNA mass 0.1%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 0.5% collodion solution by volume 0.25：1 (25%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 5% reaction water phase is made into, is heated to 30 DEG C.With styrene and diethyl The mix monomer of alkene benzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed into body Product, than being 3: 1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3For Template agent, is to slowly warm up to 95 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, peroxide It is the 2% of mix monomer gross mass, Nano-meter CaCO3 to change the consumption of benzoyl3Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour boils half an hour with 3mol/L HCl/water solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Embodiment 11 prepares the DNA immunization adsorbent that sample number into spectrum is No. 11
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 2 times of resin, stirs 10min, The ethanol that resin volume ratio is 1/1 is added after filtering, 1min is stirred, after being dried 2 hours at the resin after filtering is placed on into 60 DEG C Cooling obtains the DNA immunization adsorbent that sample number into spectrum is No. 11.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 2mg/ml.Glycine is added toward the DNA stostes for preparing, and (consumption is DNA stostes Middle DNA mass 0.1%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1% collodion solution by volume 0.25：1 (25%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 0.5% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 4: 1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour boils half an hour with 3mol/L HCl/water solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Embodiment 12 prepares the DNA immunization adsorbent that sample number into spectrum is No. 12
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 2 times of resin, stirs 5min, The ethanol ether mixed solvent that resin volume ratio is 1.5/1 is added after filtering, the concentration of volume percent of ethanol is 70%, stirring 1min, the resin after filtering is placed on cooling after being dried 2 hours at 60 DEG C and obtains the DNA immunization absorption that sample number into spectrum is No. 12 Agent.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 2mg/ml.Glycine is added toward the DNA stostes for preparing, and (consumption is DNA stostes Middle DNA mass 0.1%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1% collodion solution by volume 0.25：1 (25%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 1.0% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 1.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 3：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour boils half an hour with 3mol/L HCl/water solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Embodiment 13 prepares the DNA immunization adsorbent that sample number into spectrum is No. 13
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 2 times of resin, stirs 5min, The ethanol ether mixed solvent that resin volume ratio is 0.75/1 is added after filtering, the concentration of volume percent of ethanol is 70%, is stirred 1min is mixed, the resin after filtering is placed on cooling after being dried 2 hours at 60 DEG C and is obtained the DNA immunization absorption that sample number into spectrum is No. 13 Agent.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 1mg/ml.Serine is added toward the DNA stostes for preparing, and (consumption is DNA stostes Middle DNA mass 2.0%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1.5% collodion solution by volume 0.5：1 (50%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 3.0% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 3：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 100 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, The consumption of benzoyl peroxide is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 2 hours boils half an hour with 2mol/L HCl/water solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Embodiment 14 prepares the DNA immunization adsorbent that sample number into spectrum is No. 14
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 4 times of resin, stirs 5min, The ethanol ether mixed solvent that resin volume ratio is 0.5/1 is added after filtering, the concentration of volume percent of ethanol is 70%, stirring 1min, the resin after filtering is placed on cooling after being dried 2 hours at 60 DEG C and obtains the DNA immunization absorption that sample number into spectrum is No. 14 Agent.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 1mg/ml.Glycine is added toward the DNA stostes for preparing, and (consumption is DNA stostes Middle DNA mass 0.1%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1% collodion solution by volume 0.25：1 (25%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 0.5% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 4：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 5 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, with 80 DEG C per small When programming rate be raised to 800 DEG C, steam activation 1 hour boils half an hour with 3mol/L HCl/water solution, with distillation wash To neutral, carbide resin is obtained.
Embodiment 15 prepares the DNA immunization adsorbent that sample number into spectrum is No. 15
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 4 times of resin, stirs 5min, The ethanol ether mixed solvent that resin volume ratio is 0.25/1 is added after filtering, the concentration of volume percent of ethanol is 70%, is stirred 2min is mixed, the resin after filtering is placed on cooling after being dried 2 hours at 60 DEG C and is obtained the DNA immunization absorption that sample number into spectrum is No. 15 Agent.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 1mg/ml.Glycine is added toward the DNA stostes for preparing, and (consumption is DNA stostes Middle DNA mass 1.0%) and the absolute ethyl alcohol isometric with DNA stostes, stirring or vibrate to being completely dissolved, as DNA is molten Liquid.
By DNA solution and 1% collodion solution by volume 0.25：1 (25%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 3.0% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 3：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 0.5% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour is boiled 1 hour with 3mol/L HCl/water solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Embodiment 16 prepares the DNA immunization adsorbent that sample number into spectrum is No. 16
Measure certain volume dries carbide resin, is poured into the coating solution that volume is 4 times of resin, stirs 5min, The ethanol ether mixed solvent that resin volume ratio is 0.5/1 is added after filtering, the concentration of volume percent of ethanol is 70%, stirring 1min, the resin after filtering is placed on cooling after being dried 2 hours at 60 DEG C and obtains the DNA immunization absorption that sample number into spectrum is No. 16 Agent.
The coating liquid and carbide resin used in said process are obtained by the following method respectively.
First, the preparation of coating liquid：
Take high-purity calf thymus DNA (DNA content more than 85%) be dissolved in 0.2mol/LTris-HCL (pH=7.4) delay Rush in solution, compound concentration is the DNA stostes of 1mg/ml.Added toward the DNA stostes for preparing isometric with DNA stostes anhydrous Ethanol, stirs or vibrates to being completely dissolved, as DNA solution.
By DNA solution and 1% collodion solution by volume 0.25：1 (25%v/v) is sufficiently mixed, and obtains coating liquid.
2nd, the preparation of carbide resin
Step a prepares polystyrene resin
At normal temperatures, by Gelatin in water, 0.5% reaction water phase is made into, is heated to 30 DEG C.With styrene and two The mix monomer of Ethenylbenzene is reaction oil phase, and wherein divinylbenzene accounts for the 0.5% of mix monomer gross mass.Water phase and oil phase are pressed Volume ratio is 3：1 mixing, is initiator, the Nano-meter CaCO3 for adding particle diameter to be about 50nm to benzoyl peroxide is added in system3 It is template agent, is to slowly warm up to 95 DEG C and reacts 3 hours, through filtering, after carrying out washing treatment, prepares polystyrene resin.Wherein, mistake The consumption of BP is the 1% of mix monomer gross mass, Nano-meter CaCO33Consumption for mix monomer gross mass 1%.
Step b prepares carbide resin
The polystyrene resin for adding step a to obtain in resistance furnace, gradually heats up under nitrogen protection, every with 100 DEG C The programming rate of hour is raised to 800 DEG C, and steam activation 1 hour boils half an hour with 3mol/L HCl/water solution, uses distilled water Neutrality is washed till, carbide resin is obtained.
Test：
Using ASAP2000 specific surface areas and Porosimetry to the aperture of the DNA immunization sample of sorbent resin of No. 1-16 Detected.
DNA immunization sample of sorbent preparation method and Pore Diameter Detection result are summarized as follows shown in table 1：
The DNA immunization sample of sorbent preparation method summary sheet of table 1
From table 1 it follows that using the template of different-grain diameter (10nm, 50nm) in carbide resin carrier preparation process Agent can prepare the carbide resin containing different mesopore pore sizes；Under same particle size, Nano-meter CaCO3 is used3As template when ratio Nanometer Mg (OH)2The aperture of the carbide resin prepared during as template is high, and this is probably because at follow-up resin Nano-meter CaCO3 during reason3Decomposite CO2Cause the further expansion of mesopore pore size.
Each 2ml of DNA immunization sample of sorbent that numbering is No. 1-16 is taken, is added separately in 20ml SLE patients blood plasmas, In absorption 2 hours is shaken at 37 DEG C, the SLE patients blood plasmas detection anti-ds-DNA antibody concentration before and after absorption is taken.Detection method is Enzyme linked immunosorbent assay, kit is the new production of Shanghai section, and instrument is Shenzhen Ai Kang Uranus AE120.Numbering is No. 1-16 The performance test results of DNA immunization sample of sorbent are as shown in table 2 below
The absorption property testing result of table 2
It is analyzed with reference to the data of Tables 1 and 2, we can draw：
(1) from the point of view of the result of embodiment 1-4, maintain that other conditions are constant that nano-form is added when carbide resin is prepared Agent (Nano-meter CaCO33、Mg(OH)2) after, the performance of the DNA immunization adsorbent of preparation has a certain degree of raising, is carbonized with carrier Resin varying aperture is consistent, and with resin aperture increases, DNA immunization performance of the adsorbent is stepped up.
(2) knowable to the result of comparative example 2,5 and 6, the Nano-meter CaCO3 of 50nm is being about using particle diameter3As template system Under the premise of the unification of standby carbide resin, polar amino acid is added to improve DNA immunization adsorbent in coating liquid process for preparation The absorption property (adsorption rate brings up to 82% from 75%) of product, the absorption property difference of another aspect embodiment 5 and embodiment 6 Very little, illustrates that different types of amino acid (glycine, serine) is little to the performance impact of adsorbent product.
(3) knowable to the result of comparative example 2 and 16, the Nano-meter CaCO3 of 50nm is being about using particle diameter3As template system Under the premise of the unification of standby carbide resin, appropriate mixed solvent is added to be partly dissolved collodion again after the completion of a coating, So as to the DNA " local release " of film surface will be attached to, make the film forming again of the collodion after dissolving, fixed dna, using this The absorption property (85.6%) of immunosorbent prepared by the coating mode of " wet corrosion " is prepared higher than coating mode of use The absorption property (75.5%) of immunosorbent.This explanation can reach raising DNA and effectively inhale using the coating mode of " wet corrosion " The effect of attached rate.
(4) embodiment 5,6 and embodiment 7-15 are contrasted, the premise of polarized amino acid is added in coating liquid Under, the absorption property of the immunosorbent prepared using the coating mode of " wet corrosion " is more preferable.
Under substantially the same conditions, when ethanol content is 70%-90% in redissolving solvent, DNA immunization adsorbent is inhaled Attached rate is significantly improved, and reaches 90% or so.Ethanol content too low (60%) makes-uses ethanol, the absorption of DNA adsorbents merely It is the ethanol ether mixed solvent of 70%-90% that effect is all slightly inferior to use ethanol content.
Additionally, redissolving solvent with the volume ratio of resin at (0.5-1)/1, (90% is left for the adsorption rate of DNA immunization adsorbent It is right) can reach it is very outstanding.When the volume ratio too low (0.25/1) of solvent and resin or too high (1.5/1) is redissolved, to carrying The influence of the adsorption effect of high DNA immunosorbent does not have substantially.This explanation during " wet corrosion " coating redissolve solvent into Divide and redissolve solvent to have a major impact the absorption property side for improving DNA immunization adsorbent product with the volume ratio of resin.
(5) knowable to the result of embodiment 5,14 and 16, the immunosorbent for individually being prepared using wet corrosion coating mode Adsorptivity of the absorption property (85.6%) higher than the immunosorbent for being employed alone in coating liquid adding amino acid mode to prepare Energy (82.2%), less than the immunosorbent for taking wet corrosion coating mode simultaneously and in coating liquid prepared by addition amino acid mode Absorption property (89.9%), this explanation lifting immunosorbent aspect of performance, take wet corrosion coating mode ratio in coating liquid The effect is significant of middle addition amino acid.
In a word, the invention provides a kind of preparation method of carbide resin DNA immunity adsorbent, that is, prepare and contain nanometer mould The neutral styrene-divinylbenzene polymeric adsorbent of plate agent, template agent removing is removed by carbonization and post processing, obtains mesopore enrichment Carbide resin, prepares homogeneous DNA-collodion coating liquid, and carbide resin together coating, by the side using " wet corrosion " coating Formula, is effectively improved the effective immobilized rate and immobilized stability of DNA, adsorbent overall performance is significantly improved.

Claims (10)

  1. The preparation method of 1.DNA immunosorbent, it is characterised in that comprise the following steps：
    Step one：Adsorbent coating
    Be poured into dry carbide resin in DNA coating solution by step 1.1, stirring, filtering, drying, wherein the carbonization tree Fat is 1 with the volume ratio of the DNA coatings solution：(2-4)；
    Step 1.2 to adding organic solvent in step 1.1 products therefrom, the volume ratio of the carbide resin and the organic solvent It is 1：(0.5-1), the organic solvent is ethanol or ethanol ether mixed solvent, ethanol in the ethanol ether mixed solvent Concentration of volume percent is at least 60%；
    Step 2：By the stirring of step one products therefrom, filtering, dry, cooling, finished product DNA immunization adsorbent is obtained.
  2. 2. the preparation method of DNA immunization adsorbent according to claim 1, it is characterised in that：
    The organic solvent is ethanol ether mixed solvent, the concentration of volume percent of ethanol in the ethanol ether mixed solvent It is 70%-90%.
  3. 3. the preparation method of DNA immunization adsorbent according to claim 2, it is characterised in that：
    The method that DNA coatings solution in the step one passes through to comprise the steps of is prepared：
    Step A to concentration for added in the DNA stostes of 1-2mg/ml amino acid equivalent to the 0.1%-2% of DNA mass and with The isometric absolute ethyl alcohol of DNA stostes, stirring or vibration obtain DNA solution to being completely dissolved；
    Step B by the collodion solution of step A gained DNA solution and 0.5%-1% mass concentrations by volume (0.25-0.5)： 1 is sufficiently mixed, and obtains DNA coating solution.
  4. 4. the preparation method of DNA immunization adsorbent according to claim 3, it is characterised in that：
    Amino acid described in the step A is polar amino acid, selected from glycine, serine, threonine, tyrosine, asparagus fern ammonia One or more in acid, cysteine.
  5. 5. the preparation method of DNA immunization adsorbent according to claim 3, it is characterised in that：
    Amino acid described in the step A is glycine.
  6. 6. the preparation method of DNA immunization adsorbent according to claim 2, it is characterised in that：
    The method that DNA coatings solution in the step one passes through to comprise the steps of is prepared：
    Step A, to be added and the isometric absolute ethyl alcohol of DNA stostes in the DNA stostes of 1-2mg/ml, is stirred or vibrated to concentration To being completely dissolved, DNA solution is obtained；
    Step B by step A gained DNA solution and 0.5-1.5% collodion solutions by volume (0.25-0.5)：1 is sufficiently mixed, Obtain DNA coating solution.
  7. 7. the preparation method of the DNA immunization adsorbent according to any one of claim 1 to 6, it is characterised in that：
    Carbide resin in the step one is obtained by polymerization, in polymerization, to addition nano-particle conduct in polymerization system Template.
  8. 8. the preparation method of DNA immunization adsorbent according to claim 7, it is characterised in that：
    The method that carbide resin in the step one passes through to comprise the steps of is obtained：
    Step a prepares polystyrene resin
    Initiator, nano template are added in the mixed system that Xiang Youshui phases and oil phase are constituted, 95 DEG C -100 DEG C are to slowly warm up to Reaction 3-5 hour, through filtering, after carrying out washing treatment, preparing polystyrene resin；
    Wherein, the water is mutually for concentration is the aqueous gelatin solution of mass concentration 0.5%-5%；The oil phase is styrene and diethyl The mix monomer of alkene benzene, the divinylbenzene accounts for the 0.5-1.5% of mix monomer gross mass；Water phase and oil phase volume ratio are (3- 4)：1；The consumption of the initiator is the 1-3% of mix monomer gross mass, and it is total that the consumption of the nano template accounts for mix monomer The 0.5-1% of quality；
    The preparation of step b carbide resins
    Polystyrene resin obtained in adding step a in resistance furnace, gradually heats up, with 80-100 DEG C per small under nitrogen protection When programming rate be raised to 800-1000 DEG C, steam activation 1-2 hours；It is small 0.5-1 to be boiled with 1-3mol/L HCl/water solution When, neutrality is washed to distillation, obtain carbide resin.
  9. 9. the preparation method of DNA immunization adsorbent according to claim 7, it is characterised in that：
    The nano-particle is nano-carbonate or Nanometer hydroxide.
  10. 10. the preparation method of DNA immunization adsorbent according to claim 7, it is characterised in that：
    The nano-particle is selected from Nano-meter CaCO33、MgCO3、BaCO3、SrCO3、ZnCO3、Mg(OH)2、Bi(OH)3、Fe(OH)3、Ti (OH)3、Cd(OH)2、Co(OH)2、Cr(OH)3、Mn(OH)2In any or it is various.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4469786A (en) * 1981-01-30 1984-09-04 The Mount Sinai School Of Medicine Of The City University Of New York Detection of chemical mutagens in marker rescue assay
CN105289538A (en) * 2015-11-04 2016-02-03 珠海健帆生物科技股份有限公司 DNA immunity adsorbent and preparation method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4469786A (en) * 1981-01-30 1984-09-04 The Mount Sinai School Of Medicine Of The City University Of New York Detection of chemical mutagens in marker rescue assay
CN105289538A (en) * 2015-11-04 2016-02-03 珠海健帆生物科技股份有限公司 DNA immunity adsorbent and preparation method thereof

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