DNA virus tracer encapsulated with protein by GA covalent coupling method and its preparation method and application

Abstract

The present invention provides a DNA virus tracer wrapped with protein by GA covalent coupling method, and its preparation method and application. The protein-wrapped DNA virus tracer of the present invention is composed of an inner core, an outer shell and a protein capsid wrapped in the outer shell; the inner core is SiO2 with nucleic acid adsorbed on the surface. 2 Nanospheres, with a layer of SiO wrapped around the outside of the core 2 The outer shell is modified by amino modification on the outer shell surface, and then the protein is covalently coupled to form a capsid by GA (glutaraldehyde). The novel DNA virus tracer provided by the present invention can effectively protect the encoding DNA, which is convenient for subsequent quantitative detection and analysis; it effectively ensures that the outermost capsid of the tracer is completely composed of the selected protein, so that the surface characteristics and migration characteristics of the tracer can be closer to the target virus.

Description

Technical Field

[0001] The invention belongs to the field of environmental protection, and in particular relates to a DNA virus tracer encapsulating protein by a GA covalent coupling method, and a preparation method and application thereof. Background Art

[0002] The basic structure of a virus includes a capsid made of protein and protected nucleic acid (DNA / RNA) enclosed in the capsid. The properties of the virus's capsid protein play a decisive role in its migration properties. Tracers used as virus substitutes should also try to make the surface properties similar to those of the target virus. Generally speaking, the outermost layer should be completely protein, and the DNA / RNA should be completely wrapped in the protein capsid. It should not be exposed to the outside or pass through the protein layer.

[0003] The existing DNA virus tracer is a covalent coupling of SiO2 nanospheres with selected proteins and DNA. The protein and DNA exist simultaneously on the surface of the tracer, which is not only contrary to the surface characteristics of the virus, but more importantly, the exposed DNA is not effectively protected, making the half-life of this tracer close to the half-life of naked DNA in this environment, but much smaller than the half-life of the target virus. False negative results may occur due to the rapid decomposition of the DNA marker. Therefore, it is urgent to develop a new type of DNA virus tracer. Summary of the invention

[0004] The purpose of the present invention is to provide a DNA virus tracer encapsulating protein by a GA covalent coupling method and a preparation method and application thereof.

[0005] Existing DNA virus tracers have the following two shortcomings: (1) the exposed DNA is not effectively protected; (2) proteins and DNA exist on the surface of the tracer at the same time, which is contrary to the surface characteristics of the virus. On the outside of SiO2 nanospheres (spheres with a diameter of about 70nm) close to the size of the target virus, the carbodiimide crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) is used to covalently couple the SiO2 nanospheres with the selected proteins and DNA ( Figure 1 a).

[0006] The present invention is conceived as follows: a layer of DNA marker is first adsorbed on the SiO2 core, and then a layer of SiO2 shell is wrapped to effectively protect the DNA, and then a capsid is formed on the outermost layer by covalently coupling protein ( Figure 1 b). Specifically, firstly SiO2 nanospheres ( Figure 2 a) Functionalized with TMAPS to make its surface positively charged ( Figure 2b), then electrostatically adsorb negatively charged DNA, and then use TMAPS to neutralize the excess negative charge and use TEOS to catalyze SiO2 growth to obtain a DNA-labeled SiO2 nanosphere SiO2 (DNA (SiO2)) ( Figure 2 c). Then different methods are used to wrap the protein in the outermost layer. The present invention adopts GA covalent coupling method to amino the SiO2 (DNA (SiO2)) surface using APTEOS (3-aminopropyltriethoxysilane) ( Figure 2 e), and then GA (glutaraldehyde) is used as a cross-linking agent to covalently couple the protein on the outermost layer to form a capsid, thereby obtaining a covalently coupled monolayer protein [Protein (SiO2 (DNA (SiO2)))] ( Figure 2 f).

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a protein-coated DNA virus tracer, which is composed of an inner core, an outer shell, and a protein capsid coated in the outer shell;

[0008] The inner core is a SiO2 nanosphere with nucleic acid adsorbed on the surface. A SiO2 shell with a thickness of y is wrapped on the outer side of the inner core. The surface of the shell is amino-modified, and then GA is covalently coupled to the protein to form a capsid with a thickness of z; wherein 12nm≤y≤15nm.

[0009] The particle size of the SiO2 nanospheres of the present invention is x, 20nm≤x≤120nm, preferably x is about 40nm.

[0010] Preferably, the particle size of the DNA virus tracer is x+2y+2z, 60nm≤x+2yb+2z≤160nm, and the particle size of the tracer is preferably about 80nm. The particle size x of the SiO2 nanosphere can be controlled according to the particle size of the target virus being traced, and fine-tuned by the thickness y of the SiO2 shell and the thickness z of the protein capsid.

[0011] In the present invention, the nucleic acid may be a single-stranded or double-stranded DNA having a length of 60-120 bp and not containing a stem-loop or hairpin structure.

[0012] The protein can be a protein with properties close to those of the capsid protein of the target virus. For example, a protein with an isoelectric point close to that of the target virus surface is selected to ensure that the surface properties are close to those of the target virus. There may be many types of proteins that make up the capsid, and the main structural proteins play a key role in the surface chemical properties of the target virus.

[0013] In a specific embodiment of the present invention, when the target virus is adenovirus, the outermost layer of the DNA virus tracer can be coated with a protein having an isoelectric point close to that of the adenovirus surface, such as bovine α s1 -Casein.

[0014] In specific applications, the DNA virus tracer of the present invention is used as a substitute consistent with the physical and surface chemical properties of the target virus. According to the decay rate of the target virus, it needs to be used in combination with a bacteriophage, which provides the decay rate of the target virus.

[0015] In a second aspect, the present invention provides a method for preparing a DNA virus tracer encapsulating protein by a GA covalent coupling method, comprising the following steps:

[0016] (1) The SiO2 nanospheres are contacted with the functionalized reagent TMAPS to make the surface of the SiO2 nanospheres positively charged, and then the nucleic acids are electrostatically adsorbed to serve as the core;

[0017] (2) contacting the core with TEOS to catalyze the growth of SiO2, and forming a SiO2 shell with a thickness of y on the outside of the core, i.e., SiO2 (DNA (SiO2)) nanospheres, wherein 12nm≤y≤15nm;

[0018] (3) APTEOS is used to amino-amino the surface of the SiO2(DNA(SiO2)) nanospheres obtained in step (2), and then glutaraldehyde (GA) is used as a cross-linking agent to bring the amino-amino SiO2(DNA(SiO2)) nanospheres into contact with a protein solution, thereby forming a protein capsid with a thickness of z on the surface of the SiO2(DNA(SiO2)) nanospheres.

[0019] Furthermore, the preparation method comprises the following steps:

[0020] 1) Preparation of SiO2 core colloidal solution: SiO2 nanospheres were suspended in isopropanol to prepare a SiO2 nanosphere colloidal solution with a concentration of about 50 mg / mL;

[0021] 2) Preparation of TMAPS-functionalized SiO2 nanospheres with positive surface charge: 1 mL of SiO2 nanosphere colloidal solution was subjected to ultrasonic treatment to obtain a uniform SiO2 nanosphere colloidal solution; 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours, and washed with isopropanol to obtain SiO2 nanospheres with positive surface charge;

[0022] 3) Electrostatic adsorption of DNA and SiO2 shell packaging: first prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL ultrapure water, 320 μL nucleic acid solution and 35 μL SiO2 nanospheres with positive surface charge into a centrifuge tube, mix and then ultrasonically treat to obtain a uniform solution; wash with ultrapure water by centrifugation and then ultrasonically treat to obtain a uniform colloidal solution; add 0.5 μL TMAPS solution, mix well, then add 0.5 μL TEOS, and stir at 900 rpm for 4 hours at room temperature; then add 4 μL TEOS to the system, and stir at 900 rpm for 4 days at room temperature; wash with ultrapure water, and vacuum freeze-dry to obtain SiO2 (DNA (SiO2)) nanospheres;

[0023] 4) Preparation of aminated SiO2(DNA(SiO2)) nanospheres: 0.28 mL of APTEOS and 0.1 g of dried SiO2(DNA(SiO2)) nanospheres were mixed in toluene (in order to avoid hydrolysis of APTEOS before reaction with SiO2(DNA(SiO2)) nanospheres, the reaction was carried out in 30 mL of toluene), the mixture was sonicated for 10 minutes and then refluxed for 20 hours, the product was filtered, washed with methanol, and vacuum freeze-dried to obtain aminated SiO2(DNA(SiO2)) nanospheres;

[0024] 5) Preparation of protein solution: Prepare a protein solution with PBS buffer to obtain a protein solution with a concentration of 2 mg / mL;

[0025] 6) Covalent coupling of proteins: The experiments for preparing protein-coated SiO2(DNA(SiO2)) nanospheres were all conducted at room temperature (18-25°C). The aminated SiO2(DNA(SiO2)) nanospheres were mixed with 0.025% GA in PBS buffer for 12 hours to allow the aldehyde group of GA and the amino group (-NH2) to fully react on the surface of the SiO2(DNA(SiO2)) nanospheres; then the mixture was centrifuged and washed three times with PBS buffer for purification; finally, the GA-coated SiO2(DNA(SiO2)) nanospheres were dispersed in the protein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain the protein-coated DNA virus tracer [Protein(SiO2(DNA(SiO2)))].

[0026] Wherein, the pH of the PBS buffer is 7.2.

[0027] Preferably, the reflux temperature in step 4) is 120-130°C, preferably 120°C.

[0028] In step 3), ultrapure water is used to prepare a double-stranded DNA solution, wherein the double-stranded DNA is obtained by annealing a single-stranded DNA shown in any one of SEQ ID NOs: 1-4 and its reverse complementary strand.

[0029] In a third aspect, the present invention provides a protein-coated DNA virus tracer prepared according to the method. The particle size is (x+2y+2z), which can be controlled by the particle size x of the SiO2 nanospheres in the range of 60-160nm according to the particle size of the target virus being traced, and fine-tuned by the thickness y of the SiO2 shell and the thickness z of the protein capsid.

[0030] In a fourth aspect, the present invention provides the use of the DNA virus tracer in the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied; wherein the pathogens include viruses.

[0031] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0032] (i) The novel protein-coated DNA virus tracer provided by the present invention can achieve effective protection of the encoding DNA, facilitating subsequent quantitative detection and analysis.

[0033] (ii) Effectively ensure that the outermost capsid of the tracer is completely composed of the selected protein, so that the surface characteristics and migration characteristics of the tracer can be closer to the target virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Comparative diagram of protein-encapsulated DNA-encoded viral tracers of the prior art (a) and the present invention (b).

[0035] Figure 2 The figure is a schematic diagram of the production process of a DNA virus tracer encapsulated with protein by the GA covalent coupling method of the present invention.

[0036] Figure 3 This is a conceptual diagram of the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied using protein-coated DNA virus tracers in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0038] Experimental reagents: SiO2 nanospheres (particle size 40nm), isopropanol (purity>99.8%), trimethyl[3-(trimethoxysilyl)propyl] chloride (TMAPS; 50% (wt / wt) dissolved in methanol), double-stranded DNA obtained by annealing the single strand and its reverse complementary strand in Table 1, ultrapure water, tetraethoxysilane (TEOS, purity ≥99.0%), 3-aminopropyltriethoxysilane (APTEOS, purity ≥99.0%), glutaraldehyde (GA, concentration 0.025%), PBS buffer (pH7.2).

[0039] SiO2 nanospheres were purchased from nanoComposix (https: / / nanocomposix.com / collections / solid-silica-sp

[0040] heres / products / nanoxact-silica-nanospheres? variant=15906837954649). Cow α s1 -Casein

[0041] Purchased from Sigma-Aldrich, USA (https: / / www.sigmaaldrich.com / US / en / search / bovine-milk-casein?focus=products&page=1&perpage=30&sort=relevance&term=bovine%20milk%20casein&type=product). Deoxyribonuclease I (DNase I) was purchased from ThermoFisher Scientific (https: / / www.thermofisher.cn / order / catalog / product / EN0521).

[0042] Table 1 DNA sequences (SEQ ID NO: 1-4)

[0043]

[0044] Note: The forward primer is shown in bold underline, and the reverse primer is shown in bold italics.

[0045] Example 1 Preparation of DNA virus tracer encapsulated with protein by GA covalent coupling method

[0046] In this example, adenovirus whose nucleic acid is double-stranded DNA is used as the target virus to prepare a protein-coated DNA virus tracer.

[0047] 1. Preparation of SiO2 core colloidal solution: SiO2 nanospheres with a particle size of 40 nm were suspended in isopropanol to prepare a SiO2 nanosphere colloidal solution with a concentration of about 50 mg / mL ( Figure 2 a).

[0048] 2. Preparation of TMAPS-functionalized SiO2 nanospheres with positive surface charge: Mix the prepared SiO2 nanosphere colloidal solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 nanosphere colloidal solution. Then add 10 μL of TMAPS solution, and then stir at 900 rpm in a constant temperature box for 12 hours at room temperature. After two centrifugal washes with isopropanol, SiO2 nanospheres with positive surface charge are obtained ( Figure 2 b).

[0049] 3. Electrostatic adsorption of DNA and SiO2 shell encapsulation: Use ultrapure water to prepare a double-stranded DAP3DNA solution (SEQ ID NO: 4) with a concentration of 50 μg / mL. Add 700 μL ultrapure water, 320 μL DNA solution and 35 μL SiO2 nanospheres with positive surface charge to the centrifuge tube, mix well, and ultrasonically treat for 2 minutes to obtain a relatively uniform solution. Wash twice with ultrapure water by centrifugation and ultrasonically treat to obtain a uniform colloidal solution. Then, add 0.5 μL 50% TMAPS solution, mix well, and then add 0.5 μL TEOS. Then stir evenly at 900 rpm at room temperature for 4 hours, then add 4 μL of TEOS to the solution, and stir at 900 rpm for another 4 days at room temperature. After washing twice with ultrapure water and mixing with ultrasonic treatment, you can get a SiO2 (DNA (SiO2)) nanosphere colloidal solution ( Figure 2 c).

[0050] 4. To ensure that the particle size of SiO2(DNA(SiO2)) is uniform, the SiO2(DNA(SiO2)) nanosphere colloidal solution is vacuum freeze-dried to obtain SiO2(DNA(SiO2)) nanospheres. In a micro grinder, 1 mg of SiO2(DNA(SiO2)) nanospheres is added to 1 g of zirconium beads, and the mixture is ground with 0.1 mm diameter zirconium beads for 2 to 8 minutes. The mixture is then centrifuged and washed three times at 21,000 g. The supernatant is removed and ultrapure water is added.

[0051] 5. The size and structure of the SiO2(DNA(SiO2)) nanospheres after grinding and washing were observed using a transmission electron microscope (TEM), and the Zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The particle size of the SiO2(DNA(SiO2)) nanospheres was about 65 nm.

[0052] 6. To ensure that the DNA is completely encapsulated in the SiO2 (DNA (SiO2)) nanospheres and effectively protected, the uniformly ground SiO2 (DNA (SiO2)) nanospheres were gently mixed in a 0.2U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that were not completely encapsulated on the surface, thus obtaining SiO2 (DNA (SiO2)) nanospheres with completely protected DNA ( Figure 2 d).

[0053] 7. Use GA covalent coupling method to wrap protein in the outermost layer:

[0054] (1) Preparation of aminated SiO2(DNA(SiO2)) nanospheres: 0.28 mL of APTEOS was used to fully contact and react with 0.1 g of the dried SiO2(DNA(SiO2)) nanospheres obtained in step 6. The mixture was ultrasonically treated for 10 minutes and then refluxed at 120° C. for 20 hours. After filtration, it was washed with methanol and vacuum freeze-dried to obtain SiO2(DNA(SiO2)) nanospheres aminated with APTEOS ( Figure 2 e). In order to avoid hydrolysis of APTEOS before reacting with SiO2(DNA(SiO2)) nanospheres, the reaction was carried out in 30 mL of toluene.

[0055] (2) Preparation of protein solution: Prepare bovine α-protein in PBS buffer (pH 7.2). s1 -Casein solution to obtain a protein solution with a concentration of 2 mg / mL.

[0056] (3) Covalent coupling of proteins: Experiments for preparing protein-coated SiO2(DNA(SiO2)) nanospheres were conducted at room temperature (18-25°C). First, the amino-modified SiO2(DNA(SiO2)) nanospheres were mixed with 0.025% GA in PBS buffer for 12 hours to allow the aldehyde group of GA and the amino group (-NH2) to fully react on the surface of the SiO2(DNA(SiO2)) nanospheres. The mixture was then centrifuged and washed three times with PBS for purification. Finally, the GA-coated SiO2(DNA(SiO2)) nanospheres were dispersed in a protein solution, reacted in PBS for 12 hours, and then washed and resuspended with PBS to obtain the protein-coated DNA virus tracer [Protein(SiO2(DNA(SiO2)))]( Figure 2 f).

[0057] Protein selection: The outermost layer is wrapped with a protein close to the isoelectric point of the adenovirus surface, and the protein molecule has enough amino groups to ensure that the protein molecule has enough amino sites to react with the SiO2 (DNA (SiO2)) nanospheres wrapped with the aldehyde crosslinking agent GA. In this example, bovine α s1 -Casein (GenBank: P02662).

[0058] 8. The size and structure of the DNA virus tracer [Protein(SiO2(DNA(SiO2)))] were observed using a transmission electron microscope (TEM), and its Zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The protein-coated DNA virus tracer [Protein(SiO2(DNA(SiO2)))] prepared in this example had a particle size of about 80 nm, which is consistent with the target virus adenovirus 70-90 nm ( Figure 2 f).

[0059] The half-life of DNA in the tracer of the present invention is consistent with the half-life of DNA in the SiO2 (DNA (SiO2)) tracer, and can be stored for more than 116 years at room temperature. Figure 1 The half-life of a tracer is similar to that of naked DNA, which is less than 1 day in general water and soil environments.

[0060] 9. Quantitative detection of DNA virus tracers

[0061] The DNA virus tracer [Protein(SiO2(DNA(SiO2)))] was soaked in a pepsin solution (simulated gastric fluid) at pH 2.0 and a trypsin solution (simulated intestinal fluid) at pH 6.8 for 2 hours and 12 hours respectively to simulate the protein digestion process to remove the outermost protein shell. Then soaked in NH4F etching solution until clear and transparent without precipitation to ensure that the silica was completely dissolved. The released DNA was then purified using the QIAquick PCR purification kit. The Taqman probe method was then used for qPCR detection, and the DNA copy number concentration of the sample was obtained by comparison with the standard curve.

[0062] 10. The SiO2(DNA(SiO2)) nanospheres with uniform size and complete DNA protection obtained at the end of step 6 ( Figure 2d) Determine the correspondence between the number of tracers and the number of encapsulated DNA copies: The number of tracers is calculated from the total mass of SiO2(DNA(SiO2)) nanospheres, the density of SiO2 and the particle size of SiO2(DNA(SiO2)) measured in step 5. The number of encapsulated DNA copies is obtained by the detection method described in step 9. The relationship between the number of tracers and the number of encapsulated DNA copies can be established, that is, how many double-stranded DNAs are encapsulated in each SiO2(DNA(SiO2)) nanosphere on average.

[0063] Example 2 Application of protein-coated DNA virus tracer

[0064] Existing DNA virus tracers cannot accurately infer the migration and transformation process of the target virus using the migration and transformation process of the tracer in the water and soil medium, and are difficult to truly apply in the field where pathogenic viruses cannot be used and only virus tracers can be used. In this embodiment, the DNA virus tracer prepared in Example 1, which has physical filtration and chemical adsorption properties in the soil close to those of the target virus and a half-life much longer than that of the target virus, is used in combination with a bacteriophage as a substitute for the target virus to infer the migration and transformation process of the pathogen, thereby realizing the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied. For example, when the target virus is an adenovirus, the corresponding bacteriophage can be a crAssphage phage.

[0065] 1. Use water infiltration with a certain head on an unsaturated vertical soil column to simulate the infiltration and filtration of viruses after applying organic fertilizers in the unsaturated zone soil, and use a saturated horizontal soil column to simulate the process of viruses migrating to the drainage ditch after entering the saturated zone soil. In order to avoid the mutual influence of tracers and target viruses in the soil, such as occupying adsorption sites, a parallel soil column experiment is planned, that is, a number of completely identical soil columns are filled, and only one tracer (DNA virus tracer or bacteriophage) or target virus is put in each time together with the conservative tracer KBr (used to reverse the soil hydrodynamic parameters). The recovery rate and penetration curve obtained in the experiment are combined with the simulation of the pollutant migration and transformation model in the soil to obtain the filtration parameters and adsorption and desorption rates of DNA virus tracers and the decay rate of bacteriophages in unsaturated zone soils and saturated zone soils under different conditions. The function of the filtration parameters, adsorption and desorption rates, and decay rate of the target virus is inferred.

[0066] 2. Take water and bottom mud from several representative ditch sections at different irrigation periods, and use a 5-meter-long, 1-meter-high, and 0.6-meter-wide flume to simulate the drainage conditions of different ditch sections at different irrigation periods. All tracers (DNA virus tracers and bacteriophages), target viruses, and conservative tracer KBr (used to reverse the hydrodynamic parameters) are put into the flume together. The concentration change curves of each tracer and target virus in the overlying water at different migration distances obtained in the experiment are combined with the convection-diffusion equation that considers the decomposition of pollutants and adsorption-desorption on particles, the sedimentation of suspended particles in water, and the scouring and siltation of water on the bottom mud. The convection-diffusion parameters and adsorption-desorption rates of DNA virus tracers and the decay rate of bacteriophages are used to reverse the convection-diffusion parameters and adsorption-desorption rates and decay rates of target viruses under different irrigation periods and different ditch sections.

[0067] When migrating in the soil, the surface chemical properties of the virus tracer and the target virus directly affect their adsorption in the soil. When migrating in the drain, the virus tracer and the target virus will also adsorb and desorb on the sediment and suspended particles in the water. This adsorption and desorption process is also closely related to their surface chemical properties. Therefore, it is particularly important to ensure that the surface of the virus tracer is completely coated with proteins like the target virus.

[0068] 3. The amount of organic fertilizer and irrigation water required to grow different crops is different, and soils of different textures have different filtering effects on pathogens. Figure 3 As shown, based on the soil column and water tank experiments and simulations in the first two steps, the DNA virus tracer of the present invention is encoded with different DNA sequences to mark the farmland and time of input, and together with the bacteriophage, it is injected into different farmlands at different times along with the organic fertilizer ( Figure 3 The DNA virus tracers put into the four farmlands in the plots were encapsulated with the four different DNA sequences in Table 1, and samples were collected from nearby groundwater wells and drainage ditches to detect the concentration changes of DNA virus tracers and bacteriophages in groundwater and surface water. Then, the function obtained by the soil column and water tank experiments and simulations in the first two steps, which jointly describes the migration and transformation process of the target virus with DNA virus tracers and bacteriophages, was used to estimate the time and concentration of the target virus entering the groundwater and drainage ditches, as well as the time and concentration of the target virus migrating at different distances in the drainage ditches, to conduct spatiotemporal tracing of pathogen non-point source pollution in the agricultural area where organic fertilizers are applied, and to trace the farmlands and time periods that are prone to pathogen non-point source pollution.

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A protein-coated DNA virus tracer, characterized in that: It consists of an inner core, an outer shell, and a protein capsid wrapped in the outer shell; The inner core is a SiO2 nanosphere with nucleic acid adsorbed on the surface, and a SiO2 shell with a thickness of y is wrapped on the outer side of the inner core, the surface of the shell is amino-modified, and then GA is covalently coupled to the protein to form a capsid with a thickness of z; wherein 12 nm≤y≤15 nm; wherein GA is glutaraldehyde; The particle size of the SiO2 nanosphere is x, 20 nm≤x≤120 nm; The particle size of the DNA virus tracer is x+2y+2z, 60 nm≤x+2y+2z≤160 nm; The nucleic acid is a single-stranded or double-stranded DNA with a length of 60-120 bp and without a stem-loop or hairpin structure; The protein is a protein having properties close to those of the capsid protein of the target virus, and the protein having properties close to those of the capsid protein is a protein having a close isoelectric point.

2. A method for preparing a DNA virus tracer encapsulating protein by GA covalent coupling method, characterized in that: The following steps are involved: (1) The SiO2 nanospheres are contacted with the functionalized reagent TMAPS to make the surface of the SiO2 nanospheres positively charged, and then the nucleic acids are electrostatically adsorbed to serve as the core; (2) contacting the core with TEOS to catalyze the growth of SiO2, and forming a SiO2 shell with a thickness of y on the outside of the core, i.e., SiO2(DNA(SiO2)) nanospheres, wherein 12 nm≤y≤15 nm; (3) using APTEOS to amino-amino the surface of the SiO2(DNA(SiO2)) nanospheres obtained in step (2), and then using GA as a cross-linking agent to bring the amino-amino SiO2(DNA(SiO2)) nanospheres into contact with a protein solution, thereby forming a protein capsid with a thickness of z on the surface of the SiO2(DNA(SiO2)) nanospheres; wherein GA is glutaraldehyde; The particle size of the SiO2 nanosphere is x, 20 nm≤x≤120 nm; The nucleic acid is a single-stranded or double-stranded DNA with a length of 60-120 bp and without a stem-loop or hairpin structure; The protein is a protein that is close in nature to the capsid protein of the target virus.

3. The method according to claim 2, characterized in that The following steps are involved: 1) Preparation of SiO2 core colloidal solution: SiO2 nanospheres were suspended in isopropanol to prepare a SiO2 nanosphere colloidal solution with a concentration of 50 mg / mL; 2) Preparation of TMAPS-functionalized SiO2 nanospheres with positive surface charge: 1 mL of SiO2 nanosphere colloidal solution was treated with ultrasound to obtain a uniform SiO2 nanosphere colloidal solution; 10 µL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature oven at room temperature for 12 hours, and washed with isopropanol to obtain SiO2 nanospheres with positive surface charge; 3) Electrostatic adsorption of DNA and SiO2 shell encapsulation: First, prepare a nucleic acid solution with a concentration of 50 µg / mL with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL SiO2 nanospheres with positive surface charge into a centrifuge tube, mix and then treat by ultrasound to obtain a uniform solution; wash with ultrapure water by centrifugation and then treat by ultrasound to obtain a uniform colloidal solution; add 0.5 µL TMAPS solution, mix well, then add 0.5 µL TEOS, and stir at 900 rpm for 4 hours at room temperature; then add 4 µL TEOS to the system, and stir at 900 rpm for 4 days at room temperature; wash with ultrapure water, and vacuum freeze-dry to obtain SiO2 (DNA (SiO2)) nanospheres; 4) Preparation of aminated SiO2(DNA(SiO2)) nanospheres: 0.28 mL APTEOS was mixed with 0.1 g dried SiO2(DNA(SiO2)) nanospheres in toluene, the mixture was sonicated for 10 minutes and then refluxed for 20 hours, the product was filtered, washed with methanol, and vacuum freeze-dried to obtain aminated SiO2(DNA(SiO2)) nanospheres; 5) Preparation of protein solution: Prepare a protein solution with PBS buffer to obtain a protein solution with a concentration of 2 mg / mL; 6) Covalent coupling of proteins: The amino-modified SiO2(DNA(SiO2)) nanospheres were mixed with 0.025% GA in PBS buffer for 12 hours, and then purified by centrifugation washing 3 times with PBS buffer; finally, the GA-coated SiO2(DNA(SiO2)) nanospheres were dispersed in the protein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain the protein-coated DNA virus tracer; Wherein, the pH of the PBS buffer is 7.

2.

4. The method according to claim 3, characterized in that The reflux temperature in step 4) is 120-130°C.

5. The method according to any one of claims 2 to 4, characterized in that: In step 3), ultrapure water is used to prepare a double-stranded DNA solution, wherein the double-stranded DNA is obtained by annealing a single-stranded DNA shown in any one of SEQ ID NOs: 1-4 and its reverse complementary strand.

6. A protein-coated DNA virus tracer prepared according to the method of any one of claims 2 to 5.

7. The DNA virus tracer according to claim 6, characterized in that Its particle size is x+2y+2z, 60 nm≤x+2y+2z≤160 nm.

8. Use of the DNA virus tracer according to claim 1, 6 or 7 in the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied; in, The pathogen is a virus.

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