CN113599536A

CN113599536A - nanoparticle-rhACE-2 compound for blocking coronavirus infection target cells, preparation method and application thereof

Info

Publication number: CN113599536A
Application number: CN202110902939.7A
Authority: CN
Inventors: 褚玲玲
Original assignee: Nanjing Nake Biomaterials Co ltd
Current assignee: Nanjing Nake Biomaterials Co ltd
Priority date: 2021-08-06
Filing date: 2021-08-06
Publication date: 2021-11-05

Abstract

The invention discloses a nanoparticle-rhACE-2 compound for blocking coronavirus infection target cells, a preparation method and application thereof, wherein the preparation method comprises the following steps: s1, preparing and purifying the rhACE-2 protein; s2, marking rhACE-2 protein by biotin; s3, preparing a nanoparticle-rhACE-2 compound: and (3) washing the nanoparticles for three times by using PBS (phosphate buffer solution), specifically washing the nanoparticles by using 1ml of PBS for the first time, washing the nanoparticles twice by using PBS with the same amount as the sample, adding soluble rhACE-2 protein which is described in S2 according to different molar ratios, incubating for 30 minutes at 37 ℃ together to enable the rhACE-2 to be effectively combined to the surfaces of the nanoparticles, and then washing the nanoparticles for three times by using PBS, wherein the product is the nanoparticle-rhACE-2 compound. The invention uses the new coronavirus receptor angiotensin converting enzyme 2 as an antagonist, and uses nanoparticles as a carrier to block and neutralize virus particles so as to inhibit the infection of the virus to target cells.

Description

nanoparticle-rhACE-2 compound for blocking coronavirus infection target cells, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a nanoparticle-rhACE-2 compound for blocking coronavirus infection of target cells, a preparation method and application thereof.

Background

Coronaviruses can be divided into 4 genera of alpha, beta, gamma and delta, plus the recent outbreak of SARS-CoV-2, and there are currently 7 kinds of coronaviruses infectious to humans, and another 6 kinds are HCoV-229E and HCoV-NL63 of the alpha genus; β HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV, the latter two of which can cause more severe clinical symptoms. SARS-CoV-2 is a coronavirus of the genus Beta, a single positive-strand RNA virus that is not segmented, and according to the genome structure, SARS-CoV-2 has approximately 79.5% homology with the SARS genome in its entire sequence and both have ACE-2 as the main receptor. The new coronary pneumonia (COVID-19) caused by the new type coronavirus (SARS-CoV-2) is still spreading continuously in the global scope, the new type coronavirus is similar to the structure of the acute respiratory syndrome coronavirus (SARS-CoV) and the middle east respiratory syndrome coronavirus (MERS-CoV), is a virus of the same genus, and the clinical manifestations of patients mainly comprise viral pneumonia symptoms such as fever, dyspnea and the like, and have imaging characteristics such as double lung infiltration. The novel coronavirus shows more special transmission characteristics than the prior infectious diseases, so that the transmission speed is higher, the transmission range is wider, and the transmission risk is higher. Firstly, the new coronavirus (SARS-nCoV) has a long latent period, generally 3-7 days, and infectivity exists in the latent period, and after the latent period of partial infectors is asymptomatic, but the infectivity also exists, so that infected patients are not easy to find, and the longer the time of exposure in public environment is, the greater the probability of infection to other susceptible people is increased.

SARS-CoV-2 is combined with human Angiotensin Converting Enzyme 2 (ACE-2) receptor, and no specific medicine is clinically verified at present. Coronavirus invades into body and replicates in target cell, and the main steps include adsorption binding, fusion entering, genetic material uncoating, biosynthesis, assembling and releasing, etc. The coronavirus is a positive-strand RNA virus with the diameter of about 100nm, and coronavirus particles mainly comprise 4 structural proteins, namely Spike protein (Spike, S), Envelope protein (Membrane, M), Envelope protein (Envelope, E) and Nucleocapsid protein (N), wherein S protein trimer mediates the connection of the virus and a host receptor and plays an important role in the aspects of host tropism, virulence and the like of the virus, so that the S protein becomes the most target for researching related vaccines. The coronavirus S protein is a type I transmembrane protein, the coronavirus S proteins of different species have different sizes, and the novel coronavirus S protein comprises 1273 amino acids and is a highly glycosylated protein. The S protein forms a homotrimer precursor protein, and is cleaved into two subunits, S1 and S2, under the action of protease: the S1 subunit is responsible for receptor binding and the S2 subunit mediates membrane fusion. S1 contains two main functional domains: the N-terminal domain (NTD) and the C-terminal domain (CTD), both of which can act as receptor binding domains. The S2 subunit contains multiple domains that play different roles in the membrane fusion process. ACE-2 is used as receptor of SARS-CoV-2, its extracellular domain is connected with spike protein on SARS-CoV-2 envelope, at this time, transmembrane serine proteinase (TMPRSS2) on cell membrane surface cuts S protein into two subunits of S1 and S2 and exposes fusion peptide, so as to achieve the goal of fusing with cell membrane, releasing genetic material and making replication. However, it has been found that SARS-CoV-2 has Furin protease cleavage site, the virus has its S protein separated into different subunit by Furin protease during assembly (S1 and S2), and then released to infect other cells, and SARS-CoV lacks Furin protease recognition site, so it is possible that SARS-CoV-2 has stronger membrane-philic fusion ability and higher cell-entering efficiency due to the difference of S protein cleavage assembly mechanism, and it has been found that the binding ability of SARS-CoV-2 and ACE-2 is 10-20 times that of SARS, resulting in high infectivity of SARS-CoV-2.

The ACE-2 receptor inhibitors chloroquine (chloroquine) and hydroxychloroquine (hydroxychloroquine) have been used as candidates for treatment of COVID-19 and have been observed in clinical trials in association with each other, but since there are clinical trials showing chloroquine and hydroxychloroquine to be unprofitable for treatment of COVID-19 and have been associated with significant adverse effects, the WHO published a statement at 25.5 months to halt hydroxychloroquine as a novel coronavirus treatment in a cluster trial. In addition, the study of SARS-CoV by some researchers found that N- (2-aminoethyl) -1 azidine-ethylamine (NAAE) which is a small molecule inhibitor of ACE-2 receptor can effectively block the cell combination mediated by coronavirus S protein, and may be used as an effective anti-SARS-CoV-2 therapeutic drug. In addition, it was found in a soluble ACE-2 protein (hrsACE-2) study that it could inhibit SARS-CoV-2 infection of Vero 6 cells, but this study still requires further in vitro and in vivo studies. Although no specific drug exists, the drug designed aiming at the combination of virus S protein and receptor protein ACE-2 has higher feasibility.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a nanoparticle-rhACE-2 compound for blocking coronavirus infection of target cells, a preparation method and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows: the preparation method of the nanoparticle-rhACE-2 compound for blocking coronavirus infection of target cells comprises the following steps:

s1, preparation and purification of rhACE-2 protein: artificially synthesizing a nucleic acid sequence corresponding to an amino acid sequence from 18 to 740 of the rhACE-2, expressing a His tag protein at the C terminal for purification, expressing an Avi tag behind the His tag for biotin labeling, connecting the sequence to a eukaryotic expression vector pRP-EGFP by using restriction enzymes to construct an expression vector pRP-EGFP-hACE-2[18-740aa ]/6xHis/Avi, transfecting the vector plasmid into 293T cells by using lipofectamine-2000, changing the liquid after 8 hours, and collecting the supernatant after 48 hours; the protein was purified according to the experimental protocol of ProteinIso Ni-IDA Resin from the entire formula gold company, specifically: centrifuging 1000g of the supernatant for 10 minutes to remove cell fragments, adding a sample into a balanced chromatographic column, washing the chromatographic column with a balancing solution after the liquid is drained, and then washing the chromatographic column with an eluent to obtain a protein solution containing the recombinant human ACE-2. Obtaining high-concentration rhACE-2 protein through WB identification;

s2, biotin-labeled rhACE-2 protein: dissolving 1 mg of rhACE-2 protein in 1ml of PBS, calculating the mole number of the protein, then balancing biotin to room temperature, then adding 2 mg of biotin into 100ul of ultrapure water, adding the biotin to reach 20 times of the protein amount, leading the rhACE-2 protein to be fully biotinylated, carrying out room temperature 30 minutes, pre-washing a purification column by using 30ml of PBS, loading, purifying the protein, and storing at-80 ℃ for later use;

s3, preparing a nanoparticle-rhACE-2 compound: and (2) washing the nanoparticles with PBS for three times, specifically washing the nanoparticles with 1ml of PBS for the first time, washing the nanoparticles with PBS equivalent to the sample for the second time, adding soluble rhACE-2 protein which is described in S2 according to different molar ratios, incubating the mixture at 37 ℃ for 30 minutes, reversing the incubation and mixing the mixture uniformly every 10 minutes to ensure that the rhACE-2 can be effectively combined to the surfaces of the nanoparticles, and then washing the nanoparticles with PBS for three times, wherein the product is the nanoparticle-rhACE-2 compound.

Preferably, the gene bank number of rhACE-2 in step S1 is: AB 046569.1.

Preferably, the streptavidin and biotin binding system is a commonly used molecular binding system, other systems including a maleimide maleimid-thio-modified ligands affinity system can also be used for coupling nanoparticles to proteins, and the invention includes, but is not limited to, the streptavidin and biotin binding system for binding rhACE2 to the surface of nanoparticles to form a nanoparticle-rhACE-2 complex (NP-rhACE-2).

Preferably, the nanoparticles include, but are not limited to, gold nanoparticles, silver nanoparticles, silica nanoparticles, ferroferric oxide nanoparticles, and degradable nanomicelles.

Preferably, the diameter of the nanoparticle includes, but is not limited to, 10nm, 100nm, 200nm, 500nm, 1000nm, 2800 nm.

The invention also provides the nanoparticle-rhACE-2 compound for blocking coronavirus infection target cells, which is prepared by the preparation method.

The invention also provides application of the nanoparticle-rhACE-2 compound extracted by the method for blocking coronavirus from infecting target cells in inhibiting the target cells from being infected by the novel coronavirus.

The effect of soluble ACE-2 and different nanoparticles on cytotoxicity was first validated and found to not cause significant cell death or apoptosis.

Further, 1) selecting nanoparticles with different materials and different diameters, and after the nanoparticles are modified by streptavidin, the surface of the nanoparticles can be combined with biotin-labeled protein. The technical scheme can be adopted to combine the receptor of the coronavirus to the surface of the nanoparticle for neutralizing the virus. The soluble nano micelle is prepared by a laboratory, firstly, diltearyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000-streptavidin) marked by streptavidin is purchased, 5mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is weighed and dissolved in 1ml of DMSO, after the mixture is fully dissolved, the mixture is dripped into 2ml of ultrapure water while stirring, the mixture is placed into a 500WM dialysis bag for dialysis for 6 hours after being stirred uniformly, the dialysis bag is taken out, and then the product is treated by ultrasonic treatment for 1 minute by a probe to obtain the streptavidin modified nano micelle.

Further, 2) synthetic rhACE-2 (GenBank: AB046569.1), expressing His tag protein at C-terminal for purification, expressing Avi tag behind the His tag for biotin labeling (FIG. 1, SEQ ID NO: 1), ligating the above sequences to eukaryotic expression vector pRP-EGFP with restriction enzyme to construct expression vector pRP-EGFP-hACE2[18-740 ]/6XHis/Avi, transfecting the above vector plasmid to 293T cells with lipofectamine-2000, changing the medium after 8 hours, and collecting the supernatant after 48 hours. The protein was purified according to the experimental protocol of ProteinIso Ni-IDA Resin from the entire formula gold company, specifically: centrifuging 1000g of the supernatant for 10 minutes to remove cell fragments, adding a sample into a balanced chromatographic column, washing the chromatographic column with a balancing solution after the liquid is drained, and then washing the chromatographic column with an eluent to obtain the protein solution containing the rhACE-2. And obtaining the rhACE-2 protein with high concentration through WB identification.

Further, 3) dissolving 1 mg of rhACE-2 protein in 1ml of PBS, calculating the mole number of the protein, then balancing biotin to room temperature, then adding 2 mg of biotin into 100ul of ultra-pure water, adding the biotin to reach 20 times of the protein amount so that the rhACE-2 protein can be fully biotinylated, carrying out room temperature for 30 minutes, pre-washing a purification column with 30ml of PBS, loading, purifying the protein, and storing at-80 ℃ for later use.

Further, 4) washing the nanoparticles in 1) with PBS for three times, specifically washing the nanoparticles with 1ml of PBS for the first time, washing the nanoparticles with PBS equivalent to the sample for the second time, adding soluble ACE-2 in 3) according to different molar ratios, incubating for 30 minutes at 37 ℃ to enable the rhACE-2 to be effectively bound to the surfaces of the nanoparticles, and then washing the nanoparticles with PBS for three times, wherein the product is nanoparticle-rhACE-2 complex (NP-rhACE 2).

Further, 4) the sequence of the Luciferase gene is integrated into a pLV-CMV vector by a Gateway method to construct a vector expression plasmid of the pLV-CMV-Luciferase for subsequent detection of the virus infection effect (figure 3, sequence 2), and the psPAX2 integrated plasmid is shown in figure 2. Meanwhile, the S gene of the new coronavirus is integrated on the PRP-CMV-human-beta-globin-intron by a gateway method to replace the originally existing envelope protein VSV-G, a new envelope plasmid PRP-CMV-human-beta-globin-intron-S is constructed, the virus packaged by the envelope plasmid expressing the S protein can be used for infecting target cells expressing ACE-2 (figure 4, sequence 3), and the recombinant plasmid is verified to be completely correct by enzyme digestion identification and sequencing. The virus is packaged by lipofectamine-2000 transfection plasmid, vector expression plasmid (pLV-CMV-Luciferase), integration plasmid (pSPAX 2) and envelope plasmid (pRP-CMV-human-beta-globin-intron-S) are added into 1ml Opti-MEM according to the proportion of 4ug:2ug:1ug according to the instruction, 12ul lipofectamine-2000 is added into 1ml Opti-MEM, the two are mixed and then are stood for 15 minutes at room temperature, the mixture is added into prepared 293T cells, the solution is changed after 8 hours, the supernatant is collected after 48 hours, 5000g is centrifuged for 30 minutes after 5000 hours, the precipitate is discarded, the supernatant is virus solution, the virus titer is determined by a plaque method, and the virus solution is subpackaged at-80 ℃ for later use.

Further, 5) the nanoparticle-soluble ACE2 (NP-rhACE-2) and the virus were incubated at 37 ℃ for 30 minutes according to different concentration gradients, then the virus was added to the target cells (293T-ACE-2), the solution was changed after 12 hours, and the luciferase value was measured after 48 hours to quantify the virus-neutralizing ability of NP-rhACE 2.

Experiments prove that the invention has the following beneficial effects:

(1) the specificity is high: the invention can effectively inhibit all coronavirus using ACE-2 as receptor from infecting target cells, and has species specificity of the viruses; (2) no drug resistance: the coronavirus belongs to RNA virus, has higher mutation rate, can effectively avoid drug resistance caused by virus mutation, and can effectively inhibit virus infection as long as the virus still needs receptor protein to enter target cells; (3) the safety is high: the material related to the invention comprises human self-protein ACE-2 and nanoparticles, wherein the nanoparticles are proved to be applicable to human bodies as drug delivery systems; ACE-2 as human self protein has higher safety, so the invention has higher safety; (4) the application flexibility is high: the invention can be used for preventing the infection of the new coronavirus and treating the diseases caused by the infection of the new coronavirus, can be combined with other protective tools such as a nose plug or a mask or directly applied to the respiratory system of a human body in the form of an aerosol, and has higher flexibility in development and application; (5) strong degradation resistance: the protein medicine is easy to digest and degrade in vivo, and can be directly adsorbed on the surface of a respiratory system after being combined with the nano particles, thereby effectively avoiding degradation; (6) high efficiency; the surface of the streptavidin-modified nanoparticle can be combined with a plurality of ACE-2 molecules, and the molecules can be combined with a plurality of virus particles to form a cascade reaction, and can be efficiently combined with free virus particles.

Drawings

FIG. 1 is a map of vector pRP-EGFP-hACE-2[18-740aa ]/6XHis/Avi expressing human recombinant soluble ACE-2;

FIG. 2 is a map of the pSPAX2 expression vector;

FIG. 3 is a map of pLV-CMV-Luciferase expression vector;

FIG. 4 is a map of the pRP-CMV-human-beta-globin-intron-S expression vector;

FIG. 5 is a graph of cytotoxicity analysis of nanoparticles of various diameters and rhACE-2;

FIG. 6 is a graph of the ability of nanoparticles of various diameters to inhibit viral infection;

FIG. 7 is a graph of a concentration gradient analysis of the ability of rhACE-2 to inhibit viral infection;

FIG. 8 is a graph of an analysis of the ability of nanoparticles of various materials to inhibit viral infection;

FIG. 9 is a graph of inhibition analysis of coronavirus infection with different ratios of nanoparticles and rhACE-2;

FIG. 10 is a graph of an assay of the ability of nanoparticle-rhACE-2 complexes of various diameters to inhibit coronavirus infection;

fig. 11 is a graph of the effect of nanoparticle-rhACE-2 complexes binding to multiple viral particles to form a cascade.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example 1 plasmid construction

The nucleic acid sequence corresponding to amino acids 18 to 740 of human angiotensin converting enzyme 2 (GenBank: AB046569.1) was synthesized artificially, and His-tag protein was expressed at the C-terminus for purification and Avi-tag was expressed behind the His-tag for biotin labeling. The specific steps are that the PCR product is 100 ng; topo vector 100 ng; BP clonase 1 ml; TE buffer to 5 μ l; at 25 ℃ for 3 h; after the reaction is finished, adding proteinase K to terminate the reaction for 10 min. The ligation product was subsequently transformed into E.coli competent cells: the method comprises the following specific steps: the volume of the competent cells was 100. mu.l, and 2. mu.l of the above reaction product was added thereto, incubated on ice for 30 min, and heat-shocked at 42 ℃ for 90 sec. Incubate for 2 min on ice. Add 250. mu.l SOC medium. The cells were incubated at 37 ℃ with shaking at 225 rpm for 1h, and 100. mu.l of the product was spread on LB plates. Incubate overnight at 37 ℃. Selecting clone colony for PCR identification, culturing positive clone with shake bacteria, extracting plasmid, enzyme cutting and sequencing. The process of ligating the plasmid of interest is as follows: 10ng of the plasmid, 60 ng of pRP-EGFP vector, 1 ul of LR clonase, and 1 ul of TE buffer up to 5ul at 25 ℃ for reaction for 3h, and adding proteinase K to terminate the reaction for 10min after the reaction is finished. Transformation of competent cells of E.coli: the volume of competent cells was 100. mu.l. Add 2. mu.l of BP reaction product to the competent cells. Incubate on ice for 30 min. Heat shock at 42 ℃ for 90 sec. Incubate for 2 min on ice. Add 250. mu.l SOC medium. The cells were incubated at 37 ℃ with shaking at 225 rpm for 1h, and 100. mu.l of the product was spread on LB plates. Incubate overnight at 37 ℃. Selecting clone colonies for PCR identification, culturing positive clone mycelia for plasmid extraction, enzyme digestion and sequencing, and obtaining an expression vector pRP-EGFP-hACE-2[18-740aa ]/6xHis/Avi (figure 1, sequence 1) through verification.

By adopting the method, the Luciferase gene sequence is integrated into a pLV-CMV vector to construct a vector expression plasmid of pLV-CMV-Luciferase, and the vector expression plasmid is used for detecting the virus infection effect (figure 3, sequence 2). The psPAX2 integrative plasmid map is shown in FIG. 3. Meanwhile, the S gene of the new coronavirus is integrated on pRP-CMV-human-beta-globin-intron to replace the originally existing envelope protein VSV-G, a new envelope plasmid pRP-CMV-human-beta-globin-intron-S is constructed, the virus packaged by the envelope plasmid expressing the S protein can be used for infecting target cells expressing ACE-2 (figure 4 and sequence 3), and the recombinant plasmid is verified to be completely correct by enzyme digestion identification and sequencing.

Example 2 packaging of pseudoviruses producing New coronavirus and protein purification

About 4x10 of the previous day⁷The individual cells were plated on 10cm cell culture dishes and prepared for virus production. The virus was packaged with lipofectamine-2000 transfection plasmid, vector expression plasmid (pLV-CMV-Luciferase), integration plasmid (pSPAX 2) and envelope plasmid (PRP-CMV-human-beta-globin-intron-S) were added to 1ml of Opti-MEM at a ratio of 4ug:2ug:1ug according to the instructions, while 12ul lipofectamine-2000 was added to another tube of 1ml of Opti-MEM, which were mixed and allowed to stand at room temperature for 15 minutes, added dropwise to prepared 293T cells, replaced fresh medium after 8 hours, and harvested after 48 hoursTaking the supernatant, centrifuging for 30 minutes at 3000g, removing the precipitate, and storing the supernatant at-80 ℃. And (5) standby. Purifying ACE-2: the expression vector pRP-EGFP-hACE-2[18-740aa ] in example 1]The/6 XHis/Avi was transfected into 293T cells using lipo2000, specifically approximately 4X107 cells plated in 10cm cell culture dishes the day before, ready for protein expression. Plasmid transfection with lipofectamine-2000: adding 1ml of Opti-MEM into one tube, then adding 12ug of the plasmid, then adding 15ul of lipofectamine-2000 into another tube, standing at room temperature for 15 minutes after mixing the two, dropwise adding the mixture into prepared 293T cells, changing fresh culture medium after 8 hours, collecting supernatant after 48 hours, centrifuging for 30 minutes at 1000g, collecting supernatant, and purifying the protein by using a His tag protein purification kit in the conway century, wherein the specific steps are as follows: firstly, uniformly mixing Ni-Agarose Resin filler, adding the mixture into a chromatographic column, standing at room temperature for 10 minutes, after gel and solution are layered, opening a liquid outlet at the bottom, and slowly flowing out ethanol under the action of gravity. And then adding deionized water with 5 times of column volume into the filled column to wash the ethanol, and then balancing the column by using Binding Buffer with 10 times of column volume. Adding the supernatant into a Binding Buffer with the same volume, loading the mixture into a column, and collecting the flow-through liquid. Then washing the column by using a solution Binding Buffer with the volume 15 times of the column volume, washing off the foreign protein, finally eluting by using an iml solution Buffer, collecting the eluent, namely the purified rhACE-2 protein, and measuring the concentration for later use.

Example 3 detection of the toxicity of different concentrations of nanoparticles and soluble ACE-2 to cells

The streptavidin-coated nanoparticles are purchased from sigma, invitrogen, seiraxi biomaterial, ltd, and the streptavidin-modified soluble nanomicelles are prepared by the laboratory, and the specific steps are as follows: firstly, commercially available streptavidin-labeled distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000-streptavidin) is placed at room temperature for 30 minutes, then 5mg is weighed and dissolved in 1ml of DMSO, after the streptavidin-labeled distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is fully dissolved, the mixture is dripped into 2ml of ultrapure water while stirring, after the mixture is uniformly stirred, the mixture is placed in a 500WM dialysis bag for dialysis for 6 hours, then the dialysis bag is taken out, and then the mixture is ultrasonically treated for 1 minute by a probe, so that a product is the streptavidin-modified nano micelle, and the product is stored at 4 ℃ for later use. The 293T cells in 1X104 holes are paved into a 96-hole Cell culture plate, ferroferric oxide nanoparticles, gold nanoparticles, silver nanoparticles and soluble nano-micelles with different diameters are added in the next day, after 48 hours, the toxicity of the nanoparticles and the soluble ACE-2 to the cells is detected by using all-type gold TransDetect Cell LIVE/DEAD Viability/cytoxicity Detection Kit, and the result shows that the virus particles do not have obvious Cytotoxicity to the cells (figure 5).

Example 4 examination of the inhibitory Effect of soluble ACE-2 and nanoparticles of different diameters and materials on coronavirus

The 293T cells of 1X104 wells were plated in 96-well cell culture plates, and the viral supernatant of example 2 and the nanoparticles of example 3 were co-incubated the following day, specifically: firstly, washing nanoparticles for three times by using PBS (phosphate buffer solution), specifically adding 1lm PBS for the first time, and centrifuging for 10 minutes at 6000 g; adsorbing the ferroferric oxide nano particles by using a magnet and then absorbing the supernatant. Then adding PBS with the same amount as the sample for washing twice, and centrifuging at 6000g for 10 minutes; adsorbing the ferroferric oxide nano particles by using a magnet and then absorbing the supernatant. Adding different amounts of nanoparticles into the virus solution described in example 2, incubating at 37 ℃ for 30 minutes to fully contact the nanoparticles with the virus, then adding the nanoparticles into the cells, and measuring the luciferase value after 48 hours; meanwhile, the purified soluble ACE-2 in the example 2 and the virus supernatant in the example 2 are respectively co-incubated for 30 minutes at 37 ℃ in different concentration gradients, then the cells are added, the value of luciferase is measured after 48 hours, and the inhibition capacity of the luciferase on virus infection is detected. The specific steps of adopting the Bright-Glo Luciferase Assay System of Promega for Luciferase detection are as follows: subjecting the cell culture plate to CO₂The incubator was removed, 100ul of the above detection reagent was added to each well of the 96-well plate, incubated at 37 ℃ for 10 minutes, and then the whole liquid was transferred to a white 96-well plate and the plate was read on the machine. KnotIt was shown that soluble ACE-2 and nanoparticles of different diameters and different materials can slightly affect viral infection of target cells by up to about 10-30% (fig. 6-8).

EXAMPLE 5 preparation of NP-rhACE-2 solution

The 293T cells of 1X104 wells are paved in a 96-well cell culture plate, and the rhACE-2 in the embodiment 2 and the nano-particles in the embodiment 3 are co-incubated the next day, specifically: firstly, washing the nanoparticles for three times by PBS (phosphate buffer solution), specifically adding 1ml of PBS for the first time, centrifuging for 10 minutes at 6000g, and adsorbing the supernatant of the ferroferric oxide nanoparticles by a magnet. Then adding PBS with the same amount as the sample for washing twice, and centrifuging at 6000g for 10 minutes; adsorbing the ferroferric oxide nano particles by using a magnet and then absorbing the supernatant. Incubating streptavidin-modified nanoparticles and biotin-modified rhACE-2 at different proportions at 37 ℃ for 30 minutes to enable the nanoparticles to be fully contacted with the rhACE-2 to form a Nano-rhACE-2 compound, centrifuging 6000g for 10 minutes, adsorbing ferroferric oxide nanoparticles by using a magnet, then absorbing supernatant, adding 1ml of PBS for washing, repeating the washing for two times, and then adding PBS with the same amount as the absorbed PBS to obtain NP-rhACE-2 solution.

Example 6 testing of NP-rhACE-2 ability to inhibit viral infection

Incubating the Nano-rhACE-2 compound and the virus supernatant in the example 2 at 37 ℃ for 30 minutes, adding all virus liquid into cells of a 96-well plate, culturing at 37 ℃, changing the liquid after 24 hours, adding 100ul of complete DMEM medium, measuring the luciferase value after 48 hours, and detecting the inhibition capacity on virus infection through luciferase. The specific steps of adopting the Bright-Glo Luciferase Assay System of Promega for Luciferase detection are as follows: the cell culture plate was removed from the CO2 incubator, 100ul of the detection reagent was added to each well of a 96-well plate, incubated at 37 ℃ for 10 minutes, and then the entire liquid was transferred to a white 96-well plate and the plate was read by machine. The NP-rhACE-2 can effectively inhibit coronavirus infection, and the maximum inhibition rate can reach 80% (figure 9-figure 10).

The invention aims at the characteristic that the coronavirus spike protein needs to be combined with a receptor protein to enter a cell, and the protein is combined on the surface of a nanoparticle to neutralize virus particles so as to achieve the effect of inhibiting viruses from infecting target cells, wherein the virus comprises but is not limited to viruses taking angiotensin enzyme 2 as a receptor. The novel method related in the invention can be developed as a specific medicine for resisting coronavirus; the method of the invention can effectively inhibit the target cells from being infected by the virus, can be used for preparing medicines and medical equipment for preventing and treating diseases caused by the coronavirus, and provides a new method for treating the coronavirus.

And (4) conclusion: experiments prove that the nanoparticle-rhACE-2 compound can inhibit the infection of novel coronavirus on target cells, and is mainly reflected in that:

(1) the specificity is high: the invention can effectively inhibit all coronavirus using ACE-2 as receptor from infecting target cells, and has species specificity of the viruses; (2) no drug resistance: the coronavirus belongs to RNA virus, has higher mutation rate, can effectively avoid drug resistance caused by virus mutation, and can effectively inhibit virus infection as long as the virus still needs receptor protein to enter target cells; (3) the safety is high: the material related to the invention comprises human self-protein ACE-2 and nanoparticles, wherein the nanoparticles are proved to be applicable to human bodies as drug delivery systems; ACE-2 as human self protein has higher safety, so the invention has higher safety; (4) the application flexibility is high: the invention can be used for preventing the infection of the new coronavirus and treating the diseases caused by the infection of the new coronavirus, can be combined with other protective tools such as a nose plug or a mask or directly applied to the respiratory system of a human body in the form of an aerosol, and has higher flexibility in development and application; (5) strong degradation resistance: the protein medicine is easy to digest and degrade in vivo, and can be directly adsorbed on the surface of a respiratory system after being combined with the nano particles, thereby effectively avoiding degradation; (6) high efficiency; the surface of the streptavidin modified nanoparticle can be combined with a plurality of ACE-2 molecules, and the molecules can be combined with a plurality of virus particles to form a cascade reaction (see figure 11), and free virus particles can be efficiently combined.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto, and various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be subject to the definition of the claims.

Sequence listing

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agtccaattg gatgcctccc tgctcatttg cttggtgata tgtggggtag attttggaca 780

aatctgtact ctttgacagt tccctttgga cagaaaccaa acatagatgt tactgatgca 840

atggtggacc aggcctggga tgcacagaga atattcaagg aggccgagaa gttctttgta 900

tctgttggtc ttcctaatat gactcaagga ttctgggaaa attccatgct aacggaccca 960

ggaaatgttc agaaagcagt ctgccatccc acagcttggg acctggggaa aggcgacttc 1020

aggatcctta tgtgcacaaa ggtgacaatg gacgacttcc tgacagctca tcatgagatg 1080

gggcatattc agtatgatat ggcatatgct gcacaacctt ttctgctaag aaatggagct 1140

aatgaaggat tccatgaagc tgttggggaa atcatgtcac tttctgcagc cacacctaag 1200

catttaaaat ccattggtct tctgtcaccc gattttcaag aagacaatga aacagaaata 1260

aacttcctgc tcaaacaagc actcacgatt gttgggactc tgccatttac ttacatgtta 1320

gagaagtgga ggtggatggt ctttaaaggg gaaattccca aagaccagtg gatgaaaaag 1380

tggtgggaga tgaagcgaga gatagttggg gtggtggaac ctgtgcccca tgatgaaaca 1440

tactgtgacc ccgcatctct gttccatgtt tctaatgatt actcattcat tcgatattac 1500

acaaggaccc tttaccaatt ccagtttcaa gaagcacttt gtcaagcagc taaacatgaa 1560

ggccctctgc acaaatgtga catctcaaac tctacagaag ctggacagaa actgttcaat 1620

atgctgaggc ttggaaaatc agaaccctgg accctagcat tggaaaatgt tgtaggagca 1680

aagaacatga atgtaaggcc actgctcaac tactttgagc ccttatttac ctggctgaaa 1740

gaccagaaca agaattcttt tgtgggatgg agtaccgact ggagtccata tgcagaccaa 1800

agcatcaaag tgaggataag cctaaaatca gctcttggag atagagcata tgaatggaac 1860

gacaatgaaa tgtacctgtt ccgatcatct gttgcatatg ctatgaggca gtacttttta 1920

aaagtaaaaa atcagatgat tctttttggg gaggaggatg tgcgagtggc taatttgaaa 1980

ccaagaatct cctttaattt ctttgtcact gcacctaaaa atgtgtctga tatcattcct 2040

agaactgaag ttgaaaaggc catcaggatg tcccggagcc gtatcaatga tgctttccgt 2100

ctgaatgaca acagcctaga gtttctgggg atacagccaa cacttggacc tcctaaccag 2160

ccccctgttt cccatcacca tcaccatcac ggcctgaacg atatttttga agcgcagaaa 2220

attgaatggc atgaatag 2238

<210> 2

<211> 1653

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 2

atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct agaggatgga 60

accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120

gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180

gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240

tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300

gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360

tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420

aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480

tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540

tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ctcctctgga 600

tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660

catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720

gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780

cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840

aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 900

attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 960

aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1020

gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080

gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140

acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200

tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260

ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 1320

ttaattaaat acaaaggata ccaggtggcc cccgctgaat tggagtcgat attgttacaa 1380

caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 1440

cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500

tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560

gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620

aaggccaaga agggcggaaa gtccaaattg taa 1653

<210> 3

<211> 3831

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 3

atgttcgttt tccttgttct gttgcctctc gttagtagcc aatgcgtcaa ccttactact 60

agaacccagc tccctccagc atataccaac tctttcacca ggggcgtata ttacccggac 120

aaagtgttcc gctcaagtgt gctgcattct acgcaggacc ttttcttgcc ctttttcagt 180

aatgttactt ggtttcatgc tatccatgtg tctggaacta acggaaccaa gcgctttgac 240

aaccccgtcc tccctttcaa cgatggcgtg tacttcgctt ccacggaaaa gtcaaacata 300

attcgcggct ggatctttgg tacaacactc gactcaaaga cgcagagcct gctgatcgtt 360

aataacgcta caaatgttgt gataaaggtg tgtgaatttc agttctgcaa tgatcccttc 420

ctgggtgtgt actaccataa gaataacaag agctggatgg aatccgaatt tagggtttac 480

agttccgcta acaactgcac attcgaatac gtaagccagc catttcttat ggatcttgag 540

ggcaagcaag gaaacttcaa gaacttgagg gagttcgtgt tcaaaaatat cgacggctat 600

tttaagatat atagcaagca cactccaata aacttggtgc gcgacctgcc ccagggattc 660

tctgctctgg agcccctggt ggatctgccc attggaataa acataactcg ctttcaaaca 720

ctgctcgccc tgcatcgcag ttacctcacc cctggtgata gtagttcagg atggacagca 780

ggagccgccg catactacgt cggctacctg cagcctagga ccttcttgct gaagtacaac 840

gagaacggta caataactga cgctgtggac tgcgctctgg accctctgtc cgagacgaag 900

tgcaccctga agagctttac tgttgaaaaa ggcatttacc aaaccagcaa cttccgcgtc 960

cagccaaccg agagcatcgt cagatttccc aacattacaa atctgtgtcc cttcggcgag 1020

gtgttcaacg ccacacgctt cgcttcagtg tacgcatgga accgcaagcg catatctaac 1080

tgcgtcgcgg attattctgt cctctacaac tccgcctctt tctccacctt caagtgctac 1140

ggagtgtcac cgactaagct gaacgatctc tgctttacca acgtctacgc ggactccttc 1200

gtgataagag gtgatgaagt gagacaaata gccccaggtc agactggtaa gatcgcagat 1260

tacaactaca aattgcctga tgatttcact ggttgcgtta tcgcgtggaa ctctaataac 1320

ctcgattcta aggtcggtgg taactacaat tacctgtacc gcttgtttag gaagtcaaac 1380

ctgaagcctt tcgagaggga tatttcaacc gaaatctatc aagcgggttc aacaccgtgt 1440

aacggtgtgg aaggatttaa ctgctacttc cccctgcagt cttacggatt ccagccaacc 1500

aatggcgtgg gttaccaacc ttatcgcgtg gtggttctga gtttcgaact gttgcacgct 1560

cccgccacgg tatgcggtcc caagaagagc actaacttgg tgaagaataa gtgcgtgaat 1620

ttcaatttca atggcctcac tggaactgga gtgctgaccg aatccaataa gaagttcttg 1680

cccttccagc agttcggaag agacattgct gacacaaccg acgcggtgcg cgatcctcag 1740

actctggaga tattggacat tacaccatgt tctttcggcg gtgtgtctgt cattactccg 1800

ggcacgaata ctagcaacca ggtagccgtg ctgtaccaag gtgtgaattg cacagaggtt 1860

cccgtcgcaa ttcacgctga ccagctgacc cccacgtgga gggtttacag cactggtagt 1920

aacgtcttcc agacgagagc cggttgcttg atcggagcgg aacatgtgaa taactcctac 1980

gagtgcgaca tccccatcgg agccggtata tgcgcctctt atcagacaca aactaactca 2040

cccaggagag cccgcagtgt ggcttctcaa agcattatag catacactat gtctcttggt 2100

gccgaaaatt ccgtggccta ttctaacaat tcaatcgcca tcccaaccaa cttcacaatt 2160

agcgtgacta ccgaaatact gcctgtgagc atgacgaaaa ccagcgtaga ctgcactatg 2220

tatatctgtg gagactccac tgagtgctcc aaccttctcc tgcagtacgg tagcttctgt 2280

acccaattga accgcgccct tacaggcatc gctgttgagc aagataagaa tacccaggaa 2340

gtttttgccc aggttaagca gatatacaaa acaccgccca ttaaggactt cggaggcttc 2400

aacttctctc agatactgcc tgacccctcc aagccatcaa aacgcagctt cattgaggac 2460

ctcttgttca acaaagtgac tctggctgat gctggcttca ttaagcagta cggagattgc 2520

ctgggagata ttgctgccag ggacctcatc tgcgcccaga agtttaatgg cctgacagtc 2580

ttgcccccac ttctgacaga cgagatgatt gctcagtaca catctgccct cctcgctggc 2640

accataacat ccggatggac atttggtgct ggtgctgccc tccagattcc cttcgcaatg 2700

cagatggcgt atcgctttaa cggcatcggt gtcacacaaa acgtgttgta tgagaaccaa 2760

aagctcatcg ctaaccagtt taattctgct attggtaaga ttcaggacag cctgtcatca 2820

accgcgtctg cccttggtaa gttgcaggac gtggtgaacc agaatgctca ggctttgaat 2880

actctggtga agcaactctc ttcaaatttc ggcgctatct cttctgtgtt gaacgacatc 2940

ctgagtcgcc ttgataaggt ggaagctgaa gttcaaattg atagattgat tactggcagg 3000

ctccagtctt tgcagaccta cgttacacag cagctgatta gggcggctga aattagagct 3060

tccgccaatc tggctgcaac caagatgtcc gaatgcgtcc tgggtcagtc aaagcgcgtt 3120

gacttttgtg gtaaaggcta ccacctcatg tcatttcccc agtcagcacc tcacggagta 3180

gtgttcctcc acgtcaccta cgttccagca caggaaaaga attttaccac tgcgccggca 3240

atctgtcacg acggtaaggc acacttcccc cgcgagggcg tattcgtgtc taacggaact 3300

cattggttcg tcacacagag aaacttctat gagcctcaga tcattaccac cgacaataca 3360

tttgtgtccg gtaactgcga cgttgtgatt ggaatcgtca acaacactgt gtacgatcca 3420

cttcagccag aactggatag cttcaaggaa gaattggaca aatatttcaa aaatcacact 3480

tcacccgatg tggacctggg tgacattagt ggtatcaatg cgtccgtggt caatattcaa 3540

aaagagattg acaggctcaa cgaagtggcc aagaacctga acgaaagtct tatcgatctg 3600

caagaattgg gaaagtatga gcagtacatc aagtggccgt ggtacatttg gttgggtttt 3660

atcgccggtc tgatcgccat cgttatggtt accattatgc tttgctgcat gacgagctgt 3720

tgctcctgtc tgaagggatg ctgctcttgc ggatcatgtt gcgactacaa agaccatgac 3780

ggtgattata aagatcatga tatcgattac aaggatgacg atgacaagta a 3831

Claims

1. The preparation method of the nanoparticle-rhACE-2 compound for blocking coronavirus infection of target cells is characterized by comprising the following steps of:

s1, preparation and purification of rhACE-2 protein: artificially synthesizing a nucleic acid sequence corresponding to an amino acid sequence from 18 to 740 of the rhACE-2, expressing a His tag protein at the C terminal for purification, expressing an Avi tag behind the His tag for biotin labeling, connecting the sequence to a eukaryotic expression vector pRP-EGFP by using restriction enzymes to construct an expression vector pRP-EGFP-hACE-2[18-740aa ]/6xHis/Avi, transfecting the vector plasmid into 293T cells by using lipofectamine-2000, changing the liquid after 8 hours, and collecting the supernatant after 48 hours; the protein was purified according to the experimental protocol of ProteinIso Ni-IDA Resin from the entire formula gold company, specifically: centrifuging 1000g of the supernatant for 10 minutes to remove cell fragments, adding a sample into a balanced chromatographic column, washing the chromatographic column with a balancing solution after the liquid is drained, and then washing the chromatographic column with an eluent to obtain a protein solution containing the recombinant human ACE-2 after the liquid flows down; obtaining high-concentration rhACE-2 protein through WB identification;

2. The method of claim 1, wherein the rhACE-2 gene bank of step S1 includes the gene bank accession number of rhACE-2: AB 046569.1.

3. The method of preparing nanoparticle-rhACE-2 complex for blocking coronavirus infection in a target cell according to claim 1, further comprising: nanoparticles were coupled to the rhACE-2 protein using either a streptavidin and biotin binding system or a maleimide-thiol-modified ligands affinity system.

4. The method for preparing the nanoparticle-rhACE-2 complex for blocking coronavirus infection in a target cell according to any one of claims 1 to 3, wherein the nanoparticle comprises but is not limited to gold nanoparticle, silver nanoparticle, silica nanoparticle, ferroferric oxide nanoparticle and degradable nano micelle.

5. The method of claim 4, wherein the diameter of the nanoparticle-rhACE-2 complex is 10nm, 100nm, 200nm, 500nm, 1000nm, 2800 nm.

6. nanoparticle-rhACE-2 complex for blocking coronavirus infection of target cells, which is prepared by the preparation method of any one of claims 1 to 5.

7. Use of nanoparticle-rhACE-2 complex for blocking coronavirus infection of a target cell as claimed in claim 6 for inhibiting the infection of a target cell by a novel coronavirus.