CN110551314A - Preparation method and application of non-biological nano artificial antibody - Google Patents
Preparation method and application of non-biological nano artificial antibody Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a non-biological nano artificial antibody, wherein a plurality of functional monomers and a cross-linking agent are polymerized by different components to form a gel nano-particle artificial antibody, the affinity and the selectivity of the artificial antibody to a target object are regulated by changing the proportion of the functional monomers, a molecular imprinting technology is combined, the target object is taken as a template, the specificity and the selectivity of the nano artificial antibody to the target object can be further improved, the affinity constant K D value of the screened nano artificial antibody to the target object reaches 10 -11 M, and is equivalent to an antibody and has good selectivity.
Description
Technical Field
The invention belongs to the technical field of biological nano materials, and particularly relates to a preparation method and application of a non-biological nano artificial antibody, wherein the non-biological nano artificial antibody has high affinity and high selectivity similar to natural antibodies and immune antibodies, and can be used for replacing the natural antibodies and the immune antibodies.
Background
The unique biological activity of antibodies makes them play an important role in disease diagnosis, immune control, bioseparation, food testing, environmental testing, drug testing, antibody calibrators, enzyme immobilization, protein crystallization and other basic research. However, the antibody is mainly obtained by animal immunization, and has the defects of high cost, low preparation efficiency, long screening period, difficult changeability and preservation, immunogenicity and the like, and the monoclonal antibody also has the problem of large batch performance difference and has great limitation in practical application.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, researches the protein-flexible polymer interaction by simulating the protein-protein interaction, provides a preparation method of a non-biological nano artificial antibody based on chemical directed evolution, and synthesizes a novel non-biological nano artificial antibody with the affinity and the selectivity equivalent to those of a natural antibody.
The technical scheme is as follows:
A preparation method of the non-biological nano artificial antibody comprises the following steps: n-isopropyl acrylamide, N-tert-butyl acrylamide, sodium dodecyl sulfate, a charged functional monomer, N, N' -methylene bisacrylamide as a cross-linking agent, a target object as a template molecule, and the template molecule is eluted after polymerization in a water phase under the action of an initiator to obtain the non-biological nano artificial antibody.
preferably, the target object comprises any one or more of protein, polypeptide, amino acid, nucleic acid, medicine, antibiotic, toxin, pesticide and metal ion.
Preferably, the charged functional monomer comprises any one or more of N- (3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1-vinylimidazole, N- (3-dimethylaminopropyl) methacrylamide, acrylamide, (3-acrylamidopropyl) trimethylammonium chloride, N- (2-aminoethyl) acrylamide and sodium vinylsulfonate.
Preferably, N-isopropylacrylamide is used in an amount of 5 to 60 wt.%, N-tert-butylacrylamide is used in an amount of 5 to 50 wt.%, charged functional monomer is used in an amount of 0.5 to 20 wt.%, and N, N' -methylenebisacrylamide is used in an amount of 0.5 to 10 wt.%.
Preferably, the polymerization method is any one of inverse emulsion polymerization, precipitation polymerization, or radical polymerization.
Preferably, the initiator is one or more of ammonium persulfate, azobisisobutyronitrile and tetramethylethylenediamine.
Preferably, the polymerization reaction temperature under the nitrogen atmosphere is 25-70 ℃, and the polymerization reaction time is 3-36 h.
Preferably, the template molecule elution solution is any one of NaCl, sodium citrate, glycine or sodium dodecyl sulfate, or elution of the template molecule is achieved by dialysis, temperature change, or pH change.
Preferably, the particle size of the nanoparticle artificial antibody is 10-3000 nm.
The non-biological nano artificial antibody prepared by the method can be used for vaccine adjuvants, biological separation, food detection, environmental detection, drug detection, antibody calibrator, enzyme immobilization and protein crystallization.
The invention uses the biomembrane interference technology (BLI) to determine the affinity between the artificial antibody of the nano particles and the target object, the biosensor fixes the target object, then the nano particles to be detected are placed in a detection pool, and when the interaction between the artificial antibody and the target object occurs, the thickness of the biological layer is increased. The binding dissociation time is used as an abscissa, the increment of the signal intensity caused by the drift of the interference curve is used as an ordinate, a standard curve is drawn, and the binding affinity K between the nanoparticle artificial antibody and the target object is fittedDAnd association and dissociation rates konAnd kdis。
The technical effects obtained by the invention are as follows:
(1) The polymer artificial antibody prepared by the chemical method has higher stability, longer service life and stronger capability of resisting severe environment, and overcomes the defects of long preparation period, easy inactivation, high cost, immunogenicity and the like of the traditional biological antibody.
(2) The invention combines the nanometer technology and the biomembrane interference technology to establish a high-flux screening system for the artificial antibody of the target object, and can obtain the affinity K between the nanometer artificial antibody and the target object within 20 minutesDAnd association and dissociation rates konAnd kdisGreatly shortening the screening time.
(3) The artificial antibody prepared by the method can be repeatedly used, and the cost is greatly reduced; and the synthesis process and the regeneration process are simple.
(4) the non-biological nano artificial antibody prepared by the method has higher affinity and good selectivity, has wide application, and can be used for replacing biological antibodies to be applied to various fields of disease diagnosis and treatment, vaccine adjuvants, bioseparation, food detection, environmental detection, drug detection, antibody calibrator, enzyme immobilization, protein crystallization and the like.
Drawings
FIG. 1 is a scanning electron microscope characterization diagram of nano-artificial antibodies of different particle sizes.
Fig. 2 is a scanning electron microscope characterization picture of the nano artificial antibody applied to the immune adjuvant.
Fig. 3 is a scanning electron microscopy characterization of IgG-targeted nanoartificial antibodies.
Fig. 4 is a scanning electron microscopy characterization of the nano-artificial antibody targeting IgG of fig. 3.
Figure 5 is a scanning electron microscopy characterization of Pd-L1-targeted nanoartificial antibodies.
Fig. 6 is a scanning electron microscopy characterization of a nano-artificial antibody targeting cytochrome C.
Figure 7 is a scanning electron microscopy characterization of a nano-artificial antibody targeting DAAO.
FIG. 8 shows the enzyme activities of molecularly imprinted immobilized cytochrome C, non-imprinted immobilized cytochrome C and free cytochrome C at different pH values.
FIG. 9 shows the enzymatic activities of molecular-imprinted immobilized DAAO, non-imprinted immobilized DAAO, and free DAAO at different pH values.
Fig. 10 is a scanning electron microscopy characterization of nano-artificial antibodies for protein crystallization.
Detailed Description
In order to make the technical scheme of the present invention better understood by those skilled in the art, the following describes the preparation method and use of a non-biological nano artificial antibody provided by the present invention in detail with reference to the examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
The first embodiment is as follows: preparation and screening of non-biological nano artificial antibody
1. Design and synthesis of nanoparticles
N-isopropylacrylamide (58-X mol%), charged monomer (3-acrylamidopropyl) trimethylammonium chloride (X mol%), N-tert-butylacrylamide (35 mol%), crosslinker N, N' -methylenebisacrylamide (7 mol%) and sodium dodecylsulfonate (10mg) were dissolved in water to give a total monomer concentration of 130 mM. After the initiator was added, polymerization was carried out at 65 ℃ for 3 hours under a nitrogen atmosphere with a magnetic stirrer. The polymerized solution was purified by dialysis with an excess of pure water, and the polymer nanoparticles were obtained after freeze-drying.
2. Preliminary screening of Artificial antibodies
The protein marker is used as a target object, the molecularly imprinted polymer nanoparticle is used as a bionic antibody, the affinity between the nanoparticle artificial antibody and the protein marker is measured by utilizing a biomembrane interference technology (BLI), and the biosensor is used for fixing the protein marker in the interaction molecules to form a biomembrane layer. The nanoparticles to be tested are then placed in a detection cell and the thickness of the biolayer increases when the interaction of the artificial antibody and the protein marker occurs. On the abscissa, the bound dissociation time results from a drift of the interference curveThe increase of the signal intensity of (a) is plotted as the ordinate, a standard curve is drawn, and the binding affinity K between the nanoparticle and the protein marker is fittedDAnd association and dissociation rates konAnd kdis。
3. Method for improving artificial antibody K by molecular imprinting technologyDValue and selectivity thereof
In the preliminary screening, the protein has high K with troponinDAfter the nanoparticles are in value, in order to further increase their KDValue and selectivity, the experiment adopts a molecular imprinting method to improve the specificity and the selectivity of the artificial antibody. The synthesis of imprinted polymers (MIPs) is as follows: n-isopropylacrylamide (28 mol%), N- (3-aminopropyl) methacrylic acid (30 mol%), N-tert-butylacrylamide (35 mol%), N, N' -methylenebisacrylamide (7 mol%), sodium dodecylsulfate (10mg) and 20mg of the protein marker were dissolved in water to give a total monomer concentration of 130 mM. After the initiator was added, polymerization was carried out at 45 ℃ for 12 hours under a nitrogen atmosphere with a magnetic stirrer. Then 0.04mol of NaCl is added and the mixture is stirred continuously for 30 minutes at room temperature to elute the template, finally the polymerized solution is purified by dialysis with excessive pure water (water is changed three times per day), and after freeze drying, the molecularly imprinted polymer nano-particles are obtained, and the scanning electron microscope characterization of the nano artificial antibodies with different particle diameters is shown in figure 1.
Example two: application of nano artificial antibody in preparation of vaccine adjuvant
N-isopropylacrylamide 48 mol%, acrylamide 10 mol% and N-t-butylacrylamide 40 mol%, N, N' -methylenebisacrylamide 2 mol% and sodium dodecyl sulfate SDS (30mg) were dissolved in 50mL of water at a total monomer concentration of 65mM, and the mixed solution was filtered, purged with nitrogen for 30min, added with ammonium persulfate, and reacted at 65 ℃ for 10 hours. And eluting the template by using 1M sodium chloride solution to obtain the nano artificial antibody. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. A scanning electron micrograph of the nanoartificial antibodies is shown in FIG. 2.
Example three: application of nano artificial antibody in biological separation and purification
1. Synthesis of IgG-targeted nano artificial antibody
228.6mg of N-isopropylacrylamide, 23mg of N- (3-dimethylaminopropyl) methacrylamide and 82.6mg of N-t-butylacrylamide, 50mg of N, N' -methylenebisacrylamide and sodium dodecyl sulfate SDS (30mg) were dissolved in 50mL of water at a total monomer concentration of 65mM, while 2mg of immunoglobulin G was added, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, amine persulfate was added, and the reaction was carried out at 65 ℃ for 10 hours. And eluting the template by using 1M sodium chloride solution to obtain the nano artificial antibody of the target IgG. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. A scanning electron micrograph of the nanoartificial antibodies is shown in FIG. 3.
2. Preparation of nano artificial antibody monolithic column
First, it is necessary to bond a silane coupling agent to the inner wall of the capillary column. After the inner wall of the capillary column is washed clean by acetone and pure water in sequence, sodium hydroxide solution with the concentration of 1mol/L is pumped through the capillary column to fully and uniformly fill the capillary column, and then the capillary column is placed in a drying oven at 120 ℃. And then taking out the capillary column after the reaction is finished, and washing the sodium hydroxide in the capillary column by using pure water until the pH value of the pumped solution of the capillary column is neutral. The capillary column was then further rinsed with acetone and blown dry with nitrogen, and the capillary column was placed into an oven at 120 ℃ for thorough drying without sealing. 0.1g of silane coupling agent 3-methacryloxypropyltrimethoxysilane was dissolved in 0.9g of anhydrous toluene in a brown vial, and 1-2 mg of a polymerization inhibitor 2, 2-biphenyl-1-picrylhydrazino was added. After pumping it through a dry capillary column, the end of the capillary column was sealed and placed in an oven at 120 ℃ for reaction. Finally the capillary column was flushed with acetone and dried with nitrogen.
Then synthesizing a nanoparticle artificial antibody monolithic column, uniformly mixing 240mg of monomer glycidyl methacrylate, 160mg of coupling agent ethylene glycol dimethacrylate, 100mg of troponin I nanoparticle artificial antibody, pore-forming agents lauryl alcohol and cyclohexanol, and adding 4mg of initiator Azoisobutyronitrile (AIBN). The mixed solution was sonicated for 1 to 2 minutes and then purged with nitrogen, whereupon the mixed solution was pumped into a capillary column, sealed at both ends, and placed in a water bath at 60 ℃ for 24 hours. After the reaction was complete, the column was washed with methanol to remove the porogen.
3. Preparation of agarose gel microsphere bonded with nano artificial antibody
The agarose gel microspheres bonded with the nano artificial antibody are prepared in a 1L open reaction kettle with built condensation reflux by adopting an emulsification-solidification method. The specific operation process is as follows: 12g of agar powder is weighed, stirred and swelled in 200mL of deionized water, placed in a water bath kettle at 95 ℃, heated for 5 hours, and an aqueous phase is prepared. Adding 400L of cyclohexane and 80mL of emulsifier span 85 into a well-built and fixed 1L reaction kettle, and fully mixing and preheating the mixture under the water bath condition of 70 ℃ at the rotating speed of 500rpm to prepare an oil phase. Slowly adding the water phase agar solution into the oil phase cyclohexane while it is hot, adding 500mg of nano artificial antibody, adjusting the rotation speed to 1300rpm, and continuously emulsifying for 30 min. Stopping heating the water bath, replacing hot water with cold water to slowly cool the emulsion, and controlling for 25min to solidify into microspheres. Adding deionized water while stirring, standing for 30min, pouring the upper oil phase, and washing with water, 20% ethanol, and anhydrous ethanol in turn. The microspheres were screened using appropriate standard sieves and stored in 20% ethanol for use.
4. Separation and purification of antibody drug
The nanometer artificial antibody monolithic column and the agarose gel microspheres bonded with the nanometer artificial antibody are applied to the separation and purification of antibody drugs.
Example four: application of nano artificial antibody in food detection
The nano artificial antibody is prepared by using vancomycin as a template molecule, and the specific synthetic process is as follows: 60.3mg of N-isopropylacrylamide, 1.2mg of acrylamide, 7.2. mu.L of acrylic acid and 44.5mg of N-t-butylacrylamide, 3mg of N, N' -methylenebisacrylamide and sodium dodecyl sulfate SDS (10mg) were dissolved in 50mL of water at a total monomer concentration of 20mM, while 20mg of vancomycin was added, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, 30mg of ammonium persulfate and 15. mu.L of tetramethylethylenediamine were added, and the reaction was carried out at 35 ℃ for 18 hours. And eluting the template by using 2M sodium chloride solution to obtain the nano artificial antibody of the targeted vancomycin. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. The nano artificial antibody is applied to targeted detection of vancomycin in food.
Example five: application of nano artificial antibody in environmental detection
The method for preparing the nano artificial antibody by using the bialaphos as a template molecule comprises the following specific synthetic processes: 60.3mg of N-isopropylacrylamide, 1.2mg of acrylamide, 7.2. mu.L of acrylic acid and 44.5mg of N-t-butylacrylamide, 3mg of N, N' -methylenebisacrylamide and sodium dodecyl sulfate SDS (10mg) were dissolved in 50mL of water at a total monomer concentration of 20mM, while 20mg of bialaphos was added, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, 30mg of ammonium persulfate and 15. mu.L of tetramethylethylenediamine were added, and the reaction was carried out at 35 ℃ for 18 h. And eluting the template by using 2M sodium chloride solution to obtain the nano artificial antibody of the targeting bialaphos. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. The nano artificial antibody is applied to bialaphos in a targeted detection environment.
With Cr3+The specific synthetic process for preparing the nano artificial antibody for the template molecule is as follows: 60.3mg of N- (3-dimethylaminopropyl) methacrylamide, 1.2mg of acrylamide, 7.2. mu.L of acrylic acid and 44.5mg of N-t-butylacrylamide, 3mg of N, N' -methylenebisacrylamide and sodium dodecyl sulfate SDS (10mg) were dissolved in 50mL of water at a total monomer concentration of 20mM while adding 20mg of chromium nitrate, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, 30mg of ammonium persulfate and 15. mu.L of tetramethylethylenediamine were added, and reaction was carried out at 35 ℃ for 18 h. Eluting the template by 0.2M imidazole solution to obtain the target Cr3+The nano artificial antibody of (1). Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. Application of nano artificial antibody to Cr in targeting detection environment3+。
Example six: application of nano artificial antibody in drug detection
The nanometer artificial antibody is prepared by taking ibuprofen as a template molecule, and the specific synthetic process is as follows: 60.3mg of N-isopropylacrylamide, 1.2mg of acrylamide, 7.2. mu.L of acrylic acid and 44.5mg of N-t-butylacrylamide, 3mg of N, N' -methylenebisacrylamide and sodium dodecyl sulfate SDS (10mg) were dissolved in 50mL of water at a total monomer concentration of 20mM, while 20mg of ibuprofen was added, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, 30mg of ammonium persulfate and 15. mu.L of tetramethylethylenediamine were added, and reaction was carried out at 35 ℃ for 18 hours. And eluting the template by using 2M sodium chloride solution to obtain the nano artificial antibody targeting ibuprofen. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. The nano artificial antibody is applied to target detection of ibuprofen.
Example seven: preparation of nano artificial antibody targeting Brain Natriuretic Peptide (BNP)
60.3mg of N-isopropylacrylamide, 1.2mg of acrylamide, 7.2. mu.L of acrylic acid and 44.5mg of N-t-butylacrylamide, 3mg of N, N' -methylenebisacrylamide and sodium dodecyl sulfate SDS (10mg) were dissolved in 50mL of water at a total monomer concentration of 20mM, while 10mg of BNP was added, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, 30mg of ammonium persulfate and 15. mu.L of tetramethylethylenediamine were added, and reaction was carried out at 35 ℃ for 18 h. And eluting the template by using 2M sodium chloride solution to obtain the nano artificial antibody targeting the BNP. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. The nano artificial antibody is applied to the target detection of BNP. A scanning electron micrograph of the nano-artificial antibody targeting cytochrome C is shown in figure 4.
Example eight: application of nano artificial antibody in antibody calibrator
1. Nano artificial antibody targeting PD-L1
195mg of N-isopropylacrylamide, 21.1mg of sodium vinylsulfonate and 165.3mg of N-t-butylacrylamide, 10mg of N, N' -methylenebisacrylamide and SDS (10mg) as sodium dodecylsulfate were dissolved in 50mL of water at a total monomer concentration of 65mM, while 5mg of PD-L1 polypeptide was added, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, amine persulfate was added, and reaction was carried out at 65 ℃ for 3 hours. And eluting the template by using 2M sodium chloride solution to obtain the nano artificial antibody targeting the PD-L1. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. A scanning electron micrograph of the nano-artificial antibody targeting cytochrome C is shown in figure 5.
2. Determination of affinity constant K of natural antibody, monoclonal antibody and nano artificial antibody to PD-L1 by biomembrane interference technique (BLI)DDifferent batches of natural and monoclonal antibodies were calibrated.
Example nine: directional ordered immobilization of enzyme by nano artificial antibody
(1) Synthesis of cytochrome C-targeted nano artificial antibody
60.3mg of N-isopropylacrylamide, 1.2mg of acrylamide, 7.2. mu.L of acrylic acid and 44.5mg of N-t-butylacrylamide, 3mg of N, N' -methylenebisacrylamide and sodium dodecyl sulfate SDS (10mg) were dissolved in 50mL of water at a total monomer concentration of 20mM, while 1mg of cytochrome C was added, the mixed solution was filtered, nitrogen gas was bubbled through for 30min, 30mg of ammonium persulfate and 15. mu.L of tetramethylethylenediamine were added, and reaction was carried out at 35 ℃ for 18 h. And eluting the template by using 2M sodium chloride solution to obtain the nano artificial antibody of the targeted cytochrome C. And dialyzing the reaction mixed solution for 3 days, and then freeze-drying to obtain the targeted nano artificial antibody. A scanning electron micrograph of the nano-artificial antibody targeting cytochrome C is shown in figure 6.
(2) Nano artificial antibody immobilized cytochrome C
Weighing 4.3mg of the nano artificial antibody targeting cytochrome C, dissolving in 28mL of PBS buffer solution, adding 1mg of cytochrome C for immobilization, and storing at 4 ℃ after immobilization.
(3) Synthesis of DAAO-targeted nano artificial antibody
N-isopropylacrylamide 32mg, N-t-butylacrylamide 31mg, (3-acrylamidopropyl) trimethylammonium chloride 12.4. mu.L, N, N' -methylenebisacrylamide 2mg and cetyltrimethylammonium bromide CTAB (10mg) were dissolved in 50mL of water at a total monomer concentration of 20mM, while adding 1mg of D-amino acid oxidase, the mixed solution was filtered, purged with nitrogen for 30min, added with 30mg of ammonium persulfate and 15. mu.L of tetramethylethylenediamine, and reacted at 35 ℃ for 18 hours. And eluting the template by using 2M sodium chloride solution to obtain the DAAO-targeted nano artificial antibody. Then dialyzing the reaction mixed solution for 3 days, and then freezing and drying to obtain the nano artificial antibody. A scanning electron micrograph of the DAAO-targeted nanoartificial antibody is shown in figure 7.
(4) Nano artificial antibody immobilized DAAO
Weighing 4.3mg of the DAAO-targeting nano artificial antibody, dissolving in 28mL of PBS buffer solution, adding 1mg of DAAO for immobilization, and storing at 4 ℃.
(5) Measurement of catalytic Activity of cytochrome C
Take 4.05g of Na2HPO4·12H2O and 0.58g NaH2PO4·2H2Dissolving O in 300mL of deionized water, adjusting pH to 4.5, 5, 6, 7 and 8 respectively in PBS buffer, and adding H2O2ABTS, prepared in PBS solution at various pH to a concentration of 30mmol/L H2O215mmol/L ABTS. The immobilized molecularly imprinted enzyme, the immobilized non-imprinted enzyme and the free enzyme were dissolved in PBS of each pH to prepare a solution having a cytochrome C concentration of 3. mu. mol/L. Collecting the cytochrome C solution and 30mmol/L H2O2And 15mmol/L ABTS 1mL each, and the change in light absorption at 420nm within 2min was recorded. Obtaining an enzyme activity calculation formula by using an ABTS free radical formula y ═ 0.1844xThe results of enzyme activities of the molecularly imprinted immobilized enzyme, the non-imprinted immobilized enzyme and the free enzyme at different pH values are shown in FIG. 8.
(8) Determination of the catalytic Activity of DAAO
Take 4.05g of Na2HPO4·12H2O and 0.58g NaH2PO4·2H2Dissolving O in 300mL of deionized water, adjusting the pH to 4.5, 5, 6, 7 and 8 respectively with PBS buffer, and dissolving 9mgD-aa in 6mL of PBS with various pH values to prepare 0.15% D-aa solutions with different pH values. Preparing 2mol/L hydrochloric acid solution, dissolving 12mg DNP in 12mL of 2mol/L hydrochloric acid, keeping out of the sun, weighing 3g NaOH and dissolving in 50mL deionized water, adding 1mL of D-aa solution with different pH values into a centrifuge tube, adding 1mL of corresponding molecular imprinting immobilized enzyme, non-imprinting immobilized enzyme and free enzyme, reacting for 2min, adding 2mLHCL to terminate the reaction, adding 0.5mL of reaction liquid into 0.5mThe L DNP solution reacts for 3min, 5mLNaOH is added after 3min, and after 5min of color development, the absorption value is measured at 520 nm. By standard curve formula for pyruvic acid: the catalytic activity of the enzyme can be obtained when y is 0.0581+1.18641 x and R2 is 0.99889. The results of enzyme activities of the molecularly imprinted immobilized enzyme, the non-imprinted immobilized enzyme and the free enzyme at different pH values are shown in FIG. 9.
Example ten: application of nano artificial antibody in protein crystallization
45.3mg of N-isopropylacrylamide, 7.1mg of acrylamide and 50.86mg of N-t-butylacrylamide, 167.5mg of polyethylene glycol (molecular weight: 3350g/mol), 7.75mg of N, N' -methylenebisacrylamide and SDS (30mg) of sodium dodecylsulfate were dissolved in 50mL of water at a total monomer concentration of 20mM, the above monomer solutions were mixed, the mixed solution was filtered, 2mg of protein for crystallization was added simultaneously, nitrogen gas was bubbled for 30min, ammonium persulfate was added, and the reaction was carried out at 65 ℃ for 10 hours. Then dialyzing the reaction mixture for 3 days, and freeze-drying to obtain the nano artificial antibody for protein crystallization (figure 10).
Dissolving the protein in 20mM Tris-HCl (pH 7.5) and 150mM NaCl solution to finally prepare the protein with the concentration of 12mg/mL, adding 10mg of nano artificial antibody, and uniformly mixing. And adding the mixed solution into a 24-pore plate for protein crystallization.
The method provided by the invention can obtain a cheap and stable non-biological nano artificial antibody, and overcomes the defects of high price, poor stability, difficult obtainment, immunogenicity and the like of the traditional natural antibody and immune antibody. According to the method provided by the invention, a plurality of functional monomers and a cross-linking agent are polymerized into the gel nanoparticle artificial antibody by different components, and the affinity and selectivity of the artificial antibody to a target object are regulated and controlled by changing the proportion of the functional monomers. The specificity and the selectivity of the nano artificial antibody to the target object can be further improved by combining a molecular imprinting technology and taking the target object as a template. Affinity constant K of screened nano artificial antibody to target objectDValue of up to 10-11M, is equivalent to antibody and has good selectivity. The invention obtains the artificial antibody gel nano-particles with uniform particle size and controllable size and shape. What is needed isThe obtained nano artificial antibody can be applied to the fields of disease diagnosis and treatment, vaccine adjuvant preparation, biological separation, food detection, environmental detection, drug detection, antibody calibrator, enzyme immobilization, protein crystallization and the like.
The present invention is not limited to the above-described examples, and various changes can be made without departing from the spirit and scope of the present invention within the knowledge of those skilled in the art.
Claims (10)
1. The preparation method of the non-biological nano artificial antibody is characterized in that N-isopropyl acrylamide, N-tert-butyl acrylamide, sodium dodecyl sulfate, a charged functional monomer, N, N' -methylene bisacrylamide as a cross-linking agent, a target object as a template molecule, and the template molecule eluted after polymerization in a water phase under the action of an initiator to obtain the non-biological nano artificial antibody.
2. The method for preparing the non-biological nano artificial antibody according to claim 1, wherein the target object comprises any one or more of protein, polypeptide, amino acid, nucleic acid, drug, antibiotic, toxin, pesticide and metal ion.
3. The method for preparing the non-biological nano artificial antibody according to claim 1, wherein the charged functional monomer comprises any one or more of N- (3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1-vinylimidazole, N- (3-dimethylaminopropyl) methacrylamide, acrylamide, (3-acrylamidopropyl) trimethylammonium chloride, N- (2-aminoethyl) acrylamide and sodium vinylsulfonate.
4. The method for preparing the non-biological nano artificial antibody according to claim 1, wherein the N-isopropylacrylamide is used in an amount of 5 to 60 wt%, the N-t-butylacrylamide is used in an amount of 5 to 50 wt%, the charged functional monomer is used in an amount of 0.5 to 20 wt%, and the N, N' -methylenebisacrylamide is used in an amount of 0.5 to 10 wt%.
5. The method for preparing the abiotic nano-artificial antibody according to claim 1, wherein the method of polymerization is any one of inverse emulsion polymerization, precipitation polymerization or radical polymerization.
6. The method for preparing the non-biological nano artificial antibody according to claim 1, wherein the initiator is one or more of ammonium persulfate, azobisisobutyronitrile and tetramethylethylenediamine.
7. The method for preparing the abiotic nano artificial antibody according to claim 5, wherein the polymerization temperature is 25 to 70 ℃ and the polymerization time is 3 to 36 hours under nitrogen atmosphere.
8. The method of claim 1, wherein the elution solution of the template molecule is any one of NaCl, sodium citrate, glycine or sodium dodecyl sulfate, or the elution of the template molecule is performed by dialysis, temperature change, or pH change.
9. The method of claim 1, wherein the particle size of the nanoparticle-modified antibody is 10-3000 nm.
10. Use of non-biological nano artificial antibody prepared by the method of any one of claims 1 to 9 in preparation of vaccine adjuvant, bio-separation, food detection, environmental detection, drug detection, preparation of antibody calibrator, enzyme immobilization and protein crystallization.
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