CN115141311A - Artificial simulated antibody for resisting SARS-CoV-2 virus and its application - Google Patents
Artificial simulated antibody for resisting SARS-CoV-2 virus and its application Download PDFInfo
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Abstract
The invention discloses an artificial simulated antibody, which is hydrogel polymer nanoparticles generated by the free radical chain polymerization reaction of N-acryloyl-amino acid functional monomers and acrylamide derivatives, and belongs to the technical field of chemical medicines. The artificial simulated antibody can rapidly and specifically bind with the spike protein (S protein) Receptor Binding Domain (RBD) of a wild strain and a variant strain of a new coronavirus (SARS-CoV-2), thereby blocking the binding of SARS-CoV-2S protein and a host cell angiotensin converting enzyme2 (ACE 2) receptor, further inhibiting the toxicity of SARS-CoV-2 to host cells and the replication and proliferation in the host cells, achieving the purpose of controlling the infection and spread of SARS-CoV-2, and having wide application prospects in the aspects of SARS-CoV-2 infection treatment and epidemic situation control.
Description
Technical Field
The invention belongs to the technical field of biochemistry, relates to an artificial simulated antibody for resisting SARS-CoV-2 virus, and also relates to a preparation method and application of the artificial simulated antibody.
Background
New coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) has posed a great threat to the health and safety of all human lives. The coronavirus 2 (SARS-CoV-2) causing COVID-19, which is a syndrome of acute respiratory syndrome, is mainly composed of 4 structural proteins, namely spike protein (S), envelope protein (envelope), membrane protein (membrane) and nucleocapsid protein (nucleocapsid). Wherein the S protein comprises an N-terminal S1 subunit and a C-terminal S2 subunit, the S1 subunit comprises a Receptor Binding Domain (RBD) of about 200 amino acids responsible for binding of the virus to a host receptor, and the S2 subunit is responsible for fusion of the virus to a host cell membrane. The S protein plays a crucial role in virus attachment, infection and spread, and is an important target for developing anti-SARS-CoV-2 drugs by combining with angiotensin converting enzyme2 (ACE 2) of host cells to infect the cells.
The hydrogel polymer nanoparticles are high molecular polymers formed by linking various functional monomers through C-C bond links formed by free radical initiated polymerization, the main chain of the hydrogel polymer nanoparticles is a C-C long chain, and the side chains are hydrophobic, hydrophilic and/or charged groups carried by different functional monomers, such as acrylamide compounds and acrylic acid compounds. These groups, similar in structure and nature to the amino acid side groups in the peptide chain of protein antibodies, can also provide hydrophobic, electrostatic, hydrogen bonding, and pi-pi stacking effects. Whether the hydrogel polymer nanoparticles can realize the specific molecular recognition function or not is crucial to whether the functional monomers introduced into the side chains can provide multiple interactions highly complementary with the binding sites of target biomolecules or not. Because the variety and the proportion of the functional monomer are adjustable, the synthesized hydrogel polymer nanoparticles have wide chemical diversity. Therefore, by adjusting the species and the proportion of the functional monomers, the synthesized polymer nanoparticles have a side chain structure and surface chemical properties matched with the amino acid sequence of a certain specific region of the target biological macromolecules, and can realize the specific recognition of the target biological macromolecules. The polymer nanoparticles with similar size to biological macromolecules such as proteins have the property and function similar to antibodies, and are also called as 'plastic' antibodies or artificial simulated antibodies. The polymer nanoparticles are simple in preparation process, low in cost, stable in physicochemical property, strong in tolerance to environmental changes such as pH and temperature, high in safety and capable of replacing natural antibodies to play specific roles and functions in the field of bioscience, and biological in-vivo experiments are not needed to be carried out, and potential biotoxins such as viruses or bacteria are not introduced.
The synthetic hydrogel polymer nanoparticles can embody the biomimetic properties of biological antibodies/receptors, mainly because they mimic the specific interactions between biological macromolecules. Therefore, the research on the specific molecular recognition mechanism between antigen and antibody and between antigen and receptor has a very important reference for designing and screening the polymer nanoparticles. The SARS-CoV-2 and ACE2 compound crystal structure is analyzed by some research, and the result shows that the combining site between the two is located in the RBD area of spinous process protein, and the amino acid sequence is 333-527. In the region, molecular docking is carried out, and an amino acid functional monomer with a high docking score is obtained. The analysis of the crystal structure and the binding site of the target protein-antibody/receptor compound is beneficial to the rational selection of candidate functional monomers, so that the constructed hydrogel polymer nanoparticle library has stronger targeting property and smaller library capacity, the antibody development time is shortened, and finally the hydrogel polymer nanoparticles with good affinity are obtained.
Disclosure of Invention
The first purpose of the invention is to provide an artificial mimic antibody against SARS-CoV-2 virus, which can bind to the RBD domain in wild-type SARS-CoV-2 virus and the S protein of the present mainstream mutant strain and form a stable complex, thereby inhibiting the virus infection process. Aims to solve the problem that the prior art lacks a safe and effective artificial simulated antibody aiming at the new coronavirus.
In order to achieve the purpose, the invention provides an artificial simulation antibody which is hydrogel polymer nanoparticles generated by the free radical chain polymerization reaction of an N-acryloyl-amino acid functional monomer and an acrylamide derivative.
Wherein the amino acid is L-phenylalanine.
Wherein the acrylamide derivative consists of N-alkyl acrylamide and N, N' -methylene bisacrylamide.
Further, the alkyl group is a C1-C5 alkyl group, and specifically, the N-alkylacrylamide includes N-methylacrylamide, N-ethylacrylamide, N-N-propylacrylamide, N-isopropylacrylamide, N-N-butylacrylamide, N-t-butylacrylamide, N-sec-butylacrylamide, N-pentylacrylamide, and the like.
The second object of the present invention is to provide a method for preparing the artificial mimobody: adding an initiator into an aqueous solution of an N-acryloyl-amino acid functional monomer and an acrylamide derivative, carrying out free radical chain polymerization reaction at the reaction temperature of 50-70 ℃ in a nitrogen atmosphere, and dialyzing and freeze-drying after the reaction to obtain the hydrogel polymer nanoparticles.
Preferably, the total concentration of the N-acryloyl-amino acid functional monomer and the acrylamide derivative in the reaction system is 20-100mM, wherein the N-acryloyl-amino acid functional monomer accounts for 30-80% of the molar amount.
Preferably, the initiator is ammonium persulfate.
The third purpose of the invention is to provide the application of the artificial mimic antibody in preparing anti-SARS-CoV-2 virus medicaments.
The artificial simulated antibody can be rapidly and specifically combined with the spike protein receptor binding domains of SARS-CoV-2 wild strains and variant strains, thereby blocking the combination of the spike protein and host cell ACE2 receptor, inhibiting the virus toxicity to the host cell and the replication and proliferation in the host cell body, and achieving the purpose of controlling toxic infection and spread.
The invention achieves the following beneficial effects:
the artificial simulated antibody provided by the invention can be specifically combined with the S protein RBD structural domain of SARS-CoV-2 wild type and variant strains, and has the advantages of high affinity, large adsorption capacity and high adsorption speed. The addition of the artificial simulated antibody blocks the effective combination of a cell receptor ACE2 and SARS-CoV-2 surface spike protein, thereby inhibiting the toxicity of SARS-CoV-2 to host cells and the replication and proliferation in the host cells. In addition, the artificial mimic antibody has higher biocompatibility and better drug potential. Finally, compared with biological antibodies, the artificial simulated antibody has mild synthesis conditions, simple preparation process, low cost and good stability, and can overcome the defects of difficult development, high cost, poor environmental tolerance, easy inactivation, severe storage and reaction conditions and the like of the biological antibodies.
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FIG. 1 results of selective adsorption experiments of hydrogel polymer nanoparticles to conventional proteins.
Figure 2 results of cytotoxicity assays of hydrogel polymer nanoparticles.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to specific embodiments, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. Based on the technical principle and the conception of the invention, other equivalent alternatives obtained by a person of ordinary skill in the art without creative efforts belong to the protection scope of the invention.
Key materials: SARS-CoV-2RBD-Fitc, SARS-CoV-2 wild type (wild type) RBD, mutant (Beta) RBD, (Delta) RBD and (Omicron) RBD protein competitive inhibition neutralization kits were purchased from general biology (Wuhan) technology Co., ltd, HSA-FITC, igG-FITC and IgA-FITC were purchased from Beijing Boersy technology Co., ltd, recombinant SARS-CoV-2 wild type, beta, delta and Omicron spinous process RBD proteins and human ACE2 receptor protein were prepared according to standard procedures, 96-well black enzyme was purchased from American Kelin Co., CCK-8 kits were purchased from GlpBio, hiScript II One Stem RT-PCR SYBR Green kit was purchased from NuoZanzan biology Ltd, all SARS-CoV-2 Zheng Virus was provided by the institute of sciences of Japan institute of sciences, and Vero 5, and 3-day infected cells were harvested. All pseudovirus experiments were done in the P2 laboratory and all euvirus experiments were done in the P3 laboratory.
Example 1: synthesis of artificially simulated antibodies
The method comprises the following steps: synthesis of N-acryloyl-L-phenylalanine monomer
Dissolving 18mmol of L-phenylalanine in 15mL of aqueous solution containing 36mmol of sodium hydroxide, mechanically stirring until the L-phenylalanine is completely dissolved, transferring the solution into a three-necked bottle, stirring for 10min under the condition of ice-water bath at the temperature of 0-4 ℃, slowly and dropwise adding 18mmol of acryloyl chloride into the mixture by using a separating funnel, wherein the dropwise adding time is more than 30min, reacting for 1h under the condition of ice-water bath after the dropwise adding is finished, and continuing to react for 2h at room temperature. Adjusting the pH of the obtained product to 1-3 with 6M hydrochloric acid to obtain a white solid in the solution, carrying out vacuum filtration, recrystallizing in water, purifying to obtain the product N-acryloyl-L-phenylalanine, freeze-drying, and sealing and storing at normal temperature.
Step two: preparation of hydrogel polymer nanoparticles
In the free radical chain polymerization reaction system, the mol percent of the N-acryloyl-L-phenylalanine monomer in the total monomer is 58%, the total concentration of the monomer is 65mM, and the total volume of the solution is 50mL.
Respectively taking 1.885mmol of N-acryloyl-L-phenylalanine monomer, 1.3mmol of N-tert-butyl acrylamide monomer and 0.065mmol of N, N' -methylene bisacrylamide monomer, dissolving with a small amount of ethanol, adding water, mixing, and diluting to 50mL. A needle connected with a nitrogen cylinder is inserted into the bottom of the solution through a rubber plug, another short needle is inserted into the rubber plug and placed at the top of the round-bottom flask, and nitrogen is continuously introduced for 30min to completely expel the dissolved oxygen in the reaction solution. Then a micro-syringe is used for penetrating through the rubber plug and adding 1mL ammonium persulfate solution (30 mg/mL), nitrogen is continuously introduced for 15min, then the ventilation needle head is pulled out, the sealing film is used for sealing the rubber plug, and the reaction is carried out for 3h at 65 ℃ in the nitrogen atmosphere. The reacted hydrogel polymer nanoparticle solution was transferred to a dialysis bag and dialyzed in 3L of ultrapure water for 4 days with water changes in the morning and evening each day. After dialysis, the polymer nanoparticle solution was freeze-dried and placed in a refrigerator at 4 ℃ for future use.
The yield of the polymer was measured and calculated, the surface zeta potential was measured using a malvern Zetasizer Nano, the particle size was measured using the dynamic light scattering method (DLS), and the absolute molecular weight of the polymer was measured using Static Light Scattering (SLS). The morphology and height of the polymer were analyzed by Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) observations. The results are shown in the following table.
And (4) analyzing results: the polymer yield was 77.81%, indicating that the hydrogel polymer nanoparticles are easy to synthesize; the particle size distribution coefficient (PDI) is lower, which indicates that the particle size distribution of the nanoparticles is uniform; transmission electron microscope and atomic force microscope show that the polymer surface is cellular, which provides enough sites for combining with virus, and the particles are arranged orderly, the internal system is stable, and the nano-scale particle size makes the polymer easy to enter into the body.
Example 2: evaluation of efficacy of Artificial mimotopes
1. Study on binding performance of artificial simulated antibody to SARS-CoV-2S protein RBD (WT)
(1) Binding amount measurement
The synthesized hydrogel polymer nanoparticles are prepared into a solution with the concentration of 0.2mg/mL by using ethanol, and after the solution is uniformly mixed, a 96-hole black enzyme label plate is added into each hole with the volume of 100 mu L, and the mixture is incubated overnight at 37 ℃. A SARS-CoV-2RBD-Fitc protein solution with a concentration of 250ng/mL was prepared using a phosphate buffer solution of pH 7.4. Adding 200 mu L of protein solution into each hole, and incubating for 1h at 37 ℃ in the dark at the rotating speed of 800r/min; and (5) making background control of blank holes of the ELISA plate on protein adsorption. After the binding is finished, 180 mu L of supernatant is taken, the fluorescence intensity of the supernatant is measured by a multifunctional microplate reader, RBD-Fitc protein solutions with the concentration of 0, 31.25, 62.5, 125, 250 and 500ng/mL are prepared, and a standard curve is drawn. The concentration of RBD-Fitc protein per well was calculated from the standard curve.
The bound amount Q (mg/g) of the RBD-Fitc protein was calculated as follows:
wherein C0 (ng/mL) is the initial concentration of RBD-Fitc protein in the system; c (ng/mL) is the concentration of RBD-Fitc protein remained in the solution after combination; v (mL) is the total volume of the binding system and m (g) is the mass of hydrogel polymer nanoparticles in solution, i.e. the concentration of hydrogel polymer nanoparticles multiplied by the total volume of the system.
(2) Binding Capacity determination
0.02mg of hydrogel polymer nanoparticles are fixed at the bottom of a black 96-well enzyme label plate, RBD-Fitc protein is diluted by phosphate buffer solution with pH7.4 to the concentration of 25, 50, 100, 200, 350, 500 and 1000ng/mL respectively, 200 mu L of RBD-Fitc protein solution is added into each well, and the mixture is incubated at 37 ℃ in the dark for 1h at the rotating speed of 800r/min. And fitting the data by adopting a Langmuir fitting equation to obtain the maximum theoretical binding capacity of the hydrogel polymer nanoparticles to the RBD-Fitc protein.
The Langmuir fitting equation is:
wherein Ce (ng/mL) represents the concentration of RBD-Fitc after binding equilibrium, Q (mg/g) represents the amount of binding of the nanoparticle to RBD-Fitc, qmax (mg/g) represents the maximum amount of binding, and K (ng/mL) is the binding equilibrium constant.
(3) Binding equilibration time assay
Fixing 0.02mg of hydrogel polymer nanoparticles at the bottom of a black 96-well enzyme label plate, diluting RBD-Fitc protein to 250ng/mL by using phosphate buffer solution with pH7.4, adding 200 mu L of protein solution into each well, incubating at 37 ℃ in a dark place at a constant temperature of 800r/min, incubating for 0, 2.5, 5, 10, 15, 30, 60 and 120min, taking 180 mu L of supernatant, measuring the fluorescence intensity of the supernatant by using a multifunctional enzyme label apparatus, and measuring the time required for reaching the binding equilibrium from a kinetic curve.
(4) Binding selectivity assays
The conventional protein stock solution in blood was diluted with a phosphate buffer solution of pH7.4 to give human serum albumin (HSA-Fitc), immunoglobulin G (IgG-Fitc), immunoglobulin A (IgA), and RBD-Fitc protein solutions each at a concentration of 250 ng/mL. Binding experiments were performed in the same manner as above, and the amount of binding of hydrogel polymer nanoparticles to different proteins was calculated.
And (4) analyzing results: the maximum binding capacity of the hydrogel polymer nanoparticles to RBD-Fitc protein can reach 1035.7mg/g, and the hydrogel polymer nanoparticles have strong binding capacity, and a kinetic experiment shows that the binding balance can be achieved within 30 minutes, which indicates that the binding speed is high. The selective binding experiment shows that the copolymer has little binding capacity to other proteins and has good selectivity (figure 1).
2. Competitive inhibition experiment of artificial simulated antibody on combination of SARS-CoV-2S protein RBD and ACE2
The half inhibition concentration of the hydrogel polymer nanoparticles to RBD proteins of different strains is determined by using a SARS-CoV-2 wild strain (wild type) RBD, mutant strain (Beta) RBD, (Delta) RBD and (Omicron) RBD protein competitive inhibition neutralization kit. The kit is based on a neutralization ELISA analysis method, RBD proteins of different strains are coated on an ELISA plate in advance, then positive control, negative control and analysis samples are respectively added into micropores for reaction, ACE2 protein marked by HRP is added, the interaction between the RBD and the ACE2 is blocked by a neutralization antibody aiming at the RBD in the samples, unbound reagents are washed away, a chromogenic substrate solution is added into the micropores for color development, and the color development intensity is in inverse proportion to the concentration of the RBD neutralization antibody in the samples.
Determining the level of anti-SARS-CoV-2 neutralizing antibody in the sample by using the inhibition ratio according to the following formula:
the results show that: the hydrogel polymer nanoparticles can obviously competitively inhibit the combination of ACE2 and RBD proteins. Half inhibitory concentration (IC 50) to (wild type) RBD was 0.11nM; IC50 for (Beta) RBD 0.06nM; IC50 for (Delta) RBD is 0.12nM; the IC50 for (Omicron) RBD was 0.08nM.
3. Potency of Artificial mimobodies against pseudoviral infection with SARS-CoV-2S protein from different strains VSV-luciferase-based pseudoviral inhibition experiments were determined. Hydrogel polymer nanoparticle concentrations were formulated to 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, and 4.8nM/L with a pH7.4, 10mM phosphate buffer solution. Equal volumes of hydrogel polymer nanoparticles (150. Mu.L) were mixed with 200 strains of the TCID50 pseudovirus carrying SARS-CoV-2 wild type, variant Beta, variant Delta, and variant Omicron (Chinese Vazym) in 96-well plates, while making virus control wells (VC) and cell control wells (CC). After 1h incubation, BHK-ACE2 cells were added to a 96-well white plate. Use of luciferase Activity
The measurement was carried out by a 96-microplate luminometer (Promega, USA).
Half maximal inhibitory concentration (IC 50) of hydrogel polymer nanoparticles against pseudoviruses was calculated from non-linear fit curves using GraphPad Prism software.
And (4) analyzing results: the results show that hydrogel polymer nanoparticles can significantly inhibit the activity of pseudoviruses carrying SARS-CoV-2 wild-type, variant Beta, variant Delta, and variant Omicron S proteins at half inhibitory concentrations IC50 of 2.70nM, 1.46nM, 0.23nM, and 1.72nM, respectively.
4. Determination of inhibitory potency of artificial simulated antibody on infection of different strains of SARS-CoV-2 live virus
(1) SARS-CoV-2 live virus induced cell morphology and cytopathic effects
SARS-CoV-2 low passage samples (WIV and Delta strains) were first amplified in Vero E6 cells to prepare working stock solutions. Vero E6 cells were seeded in 96-well plates (1X 10) the day before infection 4 Cells/well). Wild strain (WIV04,500TCID50) was mixed with hydrogel polymer nanoparticles at concentrations of 0.074, 2.70 and 4.91nM for the 1h variant Delta (500 TCID)50 ) were mixed with hydrogel polymer nanoparticles at concentrations of 0.074, 0.23 and 0.57nM for 1h, respectively. A blank containing no hydrogel polymer nanoparticles was prepared by incubating the virus with an equal amount of DMEM diluent. Cells on a 96-well plate were infected with a mixture of MOI 0.01, 0.05, and 0.1 for 1 hour, washed 1 time with DMEM without fetal bovine serum, and cultured for 48 hours. Finally, cellular morphology and cytopathic effects were recorded by bright field imaging and live cell fluorescence imaging.
And (4) analyzing results: the results show that as the hydrogel polymer nanoparticle concentration increases, cell activity increases and mortality decreases. When the hydrogel polymer nanoparticle concentration is 2.7nM and 0.23nM, the hydrogel polymer nanoparticle shows effective activity enhancement effect on WIV and Delta virus, and is basically consistent with the pseudovirus experiment result.
(2) Viral RNA detection and plaque assay
Vero E6 cells were seeded on 24-well plates (1X 10) the day before infection 5 Cells/well), wild strain (WIV04,500TCID50) was mixed with hydrogel polymer nanoparticles at concentrations of 0.074, 2.70 and 4.91nM for 1h, and delta variant (500 TCID 50) was mixed with hydrogel polymer nanoparticles at concentrations of 0.074, 0.23 and 0.57nM for 1h, respectively. A blank containing no hydrogel polymer nanoparticles was prepared by incubating the virus with an equal amount of DMEM diluent. Vero E6 cells were infected with the mixture at an MOI of 0.05 for 1h, washed 1 time, cultured with DMEM without fetal bovine serum for 48h, and viral RNA was detected. Total viral RNA was extracted from the supernatant of virus-infected cells using RNA purification kit, and qRT-PCR was performed using 2019-nCoV kit (HiScript II One Step qRT-PCR SYBR Green). The supernatant of the virus mixture containing hydrogel polymer nanoparticles was placed in another 24-well plate, replaced with 1.2mL DMEM, and then 5% CO at 37 ℃% 2 And then the mixture is incubated for 96h. Finally, the cover was removed and the cells were fixed with 4% paraformaldehyde for 48h and stained with 0.25% crystal violet to reveal viral plaques.
And (4) analyzing results: viral RNA and plaque assays showed that both viral titer and plaque decreased with increasing hydrogel polymer nanoparticle concentration. The hydrogel polymer nanoparticle concentrations were 2.7nM and 0.23nM, showing potent neutralization of WIV04 and Delta virus.
Example 3: safety evaluation of hydrogel polymer nanoparticle nanobodies
1. Cytotoxicity
The cytotoxicity of hydrogel polymer nanoparticles was determined using the CCK-8 activity assay. Taking cells with logarithmic growth phase and good growth state, inoculating the cells into a 96-well culture plate at the rate of 5 multiplied by 10^3 per well, growing to 70-80% of fusion rate, preparing hydrogel polymer nanoparticles (0, 0.8, 2.0, 4.0 and 8.0 nM) with different concentrations, changing the solution 4h after administration, and continuing culturing for 48h. After incubation, cells were washed with PBS buffer, then 10 μ L CCK-8 working solution mixture was added and incubated for an additional 1.5h. Cell viability was quantified by reading the absorbance (λ =450 nm) by a microplate reader. Cell viability was calculated with reference to cell viability without any treatment.
The increment rate calculation formula is as follows:
and (4) analyzing results: the experimental results are shown in fig. 2, and the hydrogel polymer nanoparticles have no obvious cytotoxicity.
2. Toxicity in vivo
Ketamine and xylazine (100 and 5mg/kg, respectively) were dissolved in 100 μ L sterile PBS, injected intraperitoneally, and then 50 μ L of PBS-diluted hydrogel polymer nanoparticles were inoculated intranasally at a dose of 10mg/kg body weight into 5-week-old female pathogen-free mice, and 5 replicates were performed. To monitor the toxicity of hydrogel polymer nanoparticles in mice, the body weight of the mice was monitored daily over a period of one week and blood indicators were monitored on days 1, 3 and 7 and compared to control mice (inoculated intranasally with 50 μ L PBS). Liver, kidney, spleen and lung tissue sections of euthanized mice one week after nasal inoculation were stained with hematoxylin and eosin stain and pathological images were observed and captured under a microscope.
And (4) analyzing results: the change in body weight was not significantly different from the control group, indicating that the hydrogel polymer nanoparticles did not have a significant effect on the growth of mice. In H & E staining pictures of all organ sections of mice, the states of liver, spleen, lung and kidney cells of the mice which are administrated as hydrogel are not much different from those of a control group, and all blood biochemical indexes are in a normal range, which shows that the hydrogel does not generate obvious toxicity to main organs of the mice 7 days after administration and has good in vivo biocompatibility.
Example 4
In the free radical chain polymerization reaction system of this example, the mole percentage of the N-acryloyl-L-phenylalanine monomer in the total monomer is 52%, the total concentration of the monomer is 45mM, and the total volume of the solution is 50mL.
Respectively taking 1.17mmol of N-acryloyl-L-phenylalanine monomer, 1.035mmol of N-isopropyl acrylamide monomer and 0.045mmol of N, N' -methylene bisacrylamide, dissolving with a small amount of ethanol, then adding water for mixing, and fixing the volume to 50mL. 1mL of ammonium persulfate solution was added and reacted under nitrogen atmosphere at a reaction temperature of 70 ℃ for 1.5 hours, and the specific operation method was the same as in example 1.
Example 5
In the free radical chain polymerization reaction system of this example, the molar percentage of the N-acryloyl-L-phenylalanine monomer to the total monomer was 62%, the total concentration of the monomer was 73mM, and the total volume of the solution was 50mL.
Respectively taking 2.263mmol of N-acryloyl-L-phenylalanine monomer, 1.314mmol of N-ethyl acrylamide monomer and 0.073mmol of N, N '-methylene bisacrylamide, dissolving the N-acryloyl-L-phenylalanine monomer, the N-ethyl acrylamide monomer and the N, N' -methylene bisacrylamide with a small amount of ethanol, adding water, mixing, and keeping the volume to 50mL. 1mL of ammonium persulfate solution was added and reacted at 52 ℃ for 4 hours under nitrogen atmosphere, and the specific operation method was the same as example 1.
Example 6
In the free radical chain polymerization reaction system of this example, the molar percentage of the N-acryloyl-L-phenylalanine monomer to the total monomers was 56%, the total concentration of the monomers was 55mM, and the total volume of the solution was 50mL.
Respectively taking 1.54mmol of N-acryloyl-L-phenylalanine monomer, 1.155mmol of N-methacrylamide monomer and 0.055mmol of N, N' -methylene bisacrylamide, dissolving with a small amount of ethanol, adding water, mixing, and diluting to 50mL. 1mL of ammonium persulfate solution was added and reacted under nitrogen atmosphere at 60 ℃ for 3 hours, and the specific operation method was the same as in example 1.
A competitive inhibition experiment of binding of SARS-CoV-2S protein RBD and ACE2 was performed on the hydrogel polymer nanoparticles prepared in examples 4 to 6 in the same manner as in example 2, and the results showed that the half inhibitory concentration (IC 50) of example 4 to (wild type) RBD was 0.16nM; example 5 half inhibitory concentration (IC 50) on (wild type) RBD was 0.23nM; example 6 half inhibitory concentration (IC 50) against (wild type) RBD was 0.15nM, and each example had inhibitory activity against SARS-CoV-2 virus.
Claims (9)
1. An artificial mimobody, comprising: the hydrogel polymer nanoparticles are generated by the free radical chain polymerization reaction of N-acryloyl-amino acid functional monomers and acrylamide derivatives.
2. The artificial mimobody of claim 1, wherein: the amino acid is L-phenylalanine.
3. The artificial mimobody of claim 1, wherein: the acrylamide derivative consists of N-alkyl acrylamide and N, N' -methylene bisacrylamide.
4. The artificial mimobody of claim 3, wherein: the alkyl is C1-C5 alkyl.
5. A method of producing an artificial mimobody as claimed in any one of claims 1 to 4, wherein: adding an initiator into an aqueous solution of an N-acryloyl-amino acid functional monomer and an acrylamide derivative, carrying out free radical chain polymerization reaction at the reaction temperature of 50-70 ℃ in a nitrogen atmosphere, and dialyzing and freeze-drying after the reaction to obtain the hydrogel polymer nanoparticles.
6. The method of claim 5, wherein: in the reaction system, the total concentration of the N-acryloyl-amino acid functional monomer and the acrylamide derivative is 20-100mM, wherein the N-acryloyl-amino acid functional monomer accounts for 30-80% of the molar weight.
7. The method of claim 5, wherein: the initiator is ammonium persulfate.
8. Use of the artificial mimobody of any one of claims 1 to 4 for the manufacture of a medicament against the SARS-CoV-2 virus.
9. The use of claim 8, wherein: the artificial simulated antibody can rapidly and specifically bind to the spike protein receptor binding domains of SARS-CoV-2 wild strain and variant strain, thereby blocking the binding of spike protein and host cell ACE2 receptor, further inhibiting the toxicity of virus to host cell and the replication and proliferation in host cell, and achieving the purpose of controlling toxic infection and spread.
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| CN110684150A (en) * | 2019-09-06 | 2020-01-14 | 南方医科大学 | Amino acid nano hydrogel and preparation method and application thereof |
| CN112076315A (en) * | 2020-08-25 | 2020-12-15 | 中国农业科学院生物技术研究所 | Nano antigen particle fused with new coronavirus S protein and ferritin subunit, new coronavirus vaccine, and preparation method and application thereof |
| CN114106166A (en) * | 2022-01-29 | 2022-03-01 | 中国疾病预防控制中心病毒病预防控制所 | Humanized anti-neocoronavirus neutralizing antibody D2 and application thereof |
| CN114173796A (en) * | 2019-06-05 | 2022-03-11 | 佛罗里达国际大学董事会 | Biotherapeutic treatment of viral infections using biopolymer based micro/nanogels |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114173796A (en) * | 2019-06-05 | 2022-03-11 | 佛罗里达国际大学董事会 | Biotherapeutic treatment of viral infections using biopolymer based micro/nanogels |
| CN110684150A (en) * | 2019-09-06 | 2020-01-14 | 南方医科大学 | Amino acid nano hydrogel and preparation method and application thereof |
| CN112076315A (en) * | 2020-08-25 | 2020-12-15 | 中国农业科学院生物技术研究所 | Nano antigen particle fused with new coronavirus S protein and ferritin subunit, new coronavirus vaccine, and preparation method and application thereof |
| CN114106166A (en) * | 2022-01-29 | 2022-03-01 | 中国疾病预防控制中心病毒病预防控制所 | Humanized anti-neocoronavirus neutralizing antibody D2 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| ORTENSIA ILARIA PARISI ET AL.: "Design and Development of Plastic Antibodies against SARS-CoV-2 RBD based on Molecularly Imprinted Polymers that Inhibit In VitroVirus Infection" * |
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