PLGA nanosphere virus tracer and its preparation method and application

Abstract

The present invention provides a PLGA nanosphere virus tracer and a preparation method and application thereof. The tracer of the present invention is composed of an inner core and a protein capsid wrapped in an outer shell; the inner core is a PLGA nanosphere containing a DNA marker, and the outer side of the inner core is covalently coupled to a protein by EDC to form a capsid. The novel DNA virus tracer provided by the present invention can achieve effective protection of the encoded DNA, which is convenient for subsequent quantitative detection and analysis; 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; and the decay rate of the DNA virus tracer is controlled by the molecular weight of PLGA and the ratio of different LA:GA, so that it is close to the target virus.

Description

Technical Field

[0001] The invention belongs to the field of environmental protection, and in particular relates to a PLGA nanosphere virus tracer 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 PLGA nanosphere virus tracer 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, so its half-life is close to that of naked DNA, which is much smaller than the target virus, and false negative results may occur due to the rapid decomposition of the DNA marker; (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. For example, the existing DNA virus tracer is a SiO2 nanosphere (a sphere with a diameter of about 70nm) with a carbodiimide crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) covalently coupled to the selected protein and DNA on the outside of the SiO2 nanosphere (a sphere with a diameter of about 70nm) close to the size of the target virus ( Figure 1 a).

[0006] The present invention is conceived as follows: PLGA nanospheres containing DNA markers are prepared to effectively protect DNA, and proteins are covalently coupled to the PLGA nanospheres containing DNA markers by EDC to form a capsid, ensuring that the outermost layer is composed of proteins, which is similar to the surface characteristics of the virus ( Figure 1b). Specifically, a PLGA nanosphere containing a DNA marker, namely PLGA (DNA), is first prepared by a nanoprecipitation method, and then a protein is encapsulated in the outermost layer by a covalent coupling method. The present invention adopts an EDC covalent coupling method, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a cross-linking agent, and covalently couples the protein in the outermost layer to form a capsid, thereby obtaining [Protein (PLGA (DNA))] ( Figure 2 c).

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

[0008] The inner core is a PLGA nanosphere containing a DNA marker, and a capsid with a thickness of y is formed on the outer side of the inner core by covalently coupling the protein through EDC; wherein 5nm≤y≤20nm;

[0009] The particle size of the PLGA nanospheres containing DNA markers is x, 55nm≤x≤65nm, preferably x is about 60nm; wherein the molecular weight of PLGA is 75-85kDa, and the molar ratio of LA:GA is 1:1;

[0010] The particle size of the tracer is x+2y, 65nm≤x+2y≤105nm, and the preferred particle size of the tracer is about 80nm; the particle size x of the PLGA nanospheres containing DNA markers can be controlled according to the particle size of the target virus being traced, and fine-tuned by the thickness y of the protein capsid.

[0011] In the present invention, 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.

[0012] The protein is a protein with properties close to the capsid protein of the target virus, and there are enough amino groups in the protein molecule to ensure that the protein molecule has enough amino sites to form covalent bonds with the carboxyl groups on the surface of the PLGA (DNA) nanospheres by dehydration condensation. For example, a protein with an isoelectric point close to the isoelectric point of the target virus surface is selected to ensure that the surface properties are close to those of the target virus.

[0013] In a specific embodiment of the present invention, when the target virus is adenovirus, the outermost layer of the tracer can be coated with a protein having a surface isoelectric point close to that of the adenovirus and a half-life greater than or equal to that of the target virus, such as human serum albumin.

[0014] In specific applications, the tracer of the present invention can be used to control the release rate of the DNA marker, that is, the decay rate of the PLGA nanosphere virus tracer, according to the decay rate of the target virus by selecting PLGA with different molecular weights and different LA:GA ratios to prepare the DNA-encapsulated core, thereby simulating the half-life of the target virus in a variety of water and soil environments.

[0015] In a second aspect, the present invention provides a method for preparing a PLGA nanosphere virus tracer having a protein capsid and containing a DNA marker, comprising the following steps:

[0016] (1) After mixing PLGA, DMSO and DNA solution, the mixture is injected into TPGS solution by a syringe pump, and the resulting emulsion is stirred in a fume hood for 4 to 8 hours (or overnight) to evaporate DMSO and harden PLGA particles, thereby preparing PLGA nanospheres containing DNA markers as the core, with a particle size x, 55 nm ≤ x ≤ 65 nm;

[0017] Among them, the molecular weight of PLGA is 75-85kDa, and the molar ratio of LA:GA is 1:1.

[0018] (2) enzymatically hydrolyzing the DNA that was not completely wrapped outside the inner core, washing and resuspending the inner core with MES buffer at pH 6.0 to obtain a PLGA (DNA) nanosphere solution with a concentration of 10 mg / mL;

[0019] (3) Using EDC as a cross-linking agent, the carboxyl groups on the outside of the PLGA (DNA) nanospheres are brought into contact with the protein solution, and a protein capsid with a thickness of y is formed on the surface of the PLGA (DNA) nanospheres through covalent coupling with EDC to obtain [Protein (PLGA (DNA))]; wherein 5nm≤y≤20nm.

[0020] Furthermore, the preparation method comprises the following steps:

[0021] 1) Preparation of DNA solution: Prepare DNA solution with ultrapure water to obtain a concentration of 4×10 -6 M DNA solution;

[0022] 2) Preparation of DNA-labeled PLGA nanospheres: 20 mg of PLGA was mixed with 1 mL of DMSO and dissolved overnight to obtain a PLGA / DMSO mixture; 5 μL of the prepared DNA solution was added to the PLGA / DMSO mixture and sonicated on ice for 10 s (40% amplitude) to obtain a DNA / PLGA / DMSO mixture; on a magnetic stirrer, the DNA / PLGA / DMSO mixture was injected into 20 mL of a 0.3 w / v% TPGS solution using a syringe pump while stirring (the tip of the syringe was below the liquid surface of the solution), and the resulting emulsion was stirred in a fume hood for 4 to 8 hours (or overnight) to evaporate the DMSO and harden the PLGA particles to obtain a solution of hardened PLGA particles;

[0023] 3) Enzymatic hydrolysis of DNA that is not completely wrapped outside the PLGA nanospheres containing DNA markers: contacting the solution of hardened PLGA particles with a DNA enzyme reagent to enzymatically hydrolyze the DNA exposed outside the nanospheres to obtain a PLGA nanosphere solution containing DNA markers, i.e., a PLGA (DNA) nanosphere solution;

[0024] 4) Preparation of protein solution: Prepare a protein solution with PBS buffer to obtain a protein solution with a concentration of 2 mg / mL;

[0025] 5) Covalent coupling of proteins: The PLGA (DNA) nanosphere solution was washed twice in MES buffer; after the second wash, the nanosphere precipitate was resuspended in 10 mL of MES buffer to obtain a PLGA (DNA) nanosphere solution with a concentration of 10 mg / mL; 10 mL of 10 mg / mL EDC was added dropwise while stirring at 18°C-25°C, and stirring was continued for 15 minutes; the solution was washed twice with PBS buffer, and then the nanospheres were resuspended with 5 mL of PBS buffer; 1.85-18.5 mL of the protein solution was added thereto, and the solution was reacted at 18°C-25°C for 2-4 hours with continuous stirring. After the reaction was completed, the solution was centrifuged and the supernatant was discarded, and the nanosphere precipitate was resuspended in 10 mL of quenching solution. After stirring for 30 minutes, the solution was centrifuged again and the supernatant was discarded, and the nanosphere precipitate was resuspended in storage buffer to obtain a PLGA nanosphere virus tracer [Protein (PLGA (DNA))], i.e., a PLGA nanosphere virus tracer having a protein capsid and containing a DNA marker.

[0026] Wherein, the pH of the MES buffer is 6.0, and the pH of the PBS buffer is 7.4;

[0027] The quenching solution is a 1 mg / mL casein solution;

[0028] The storage buffer was a 10 mg / mL glycine solution.

[0029] Unless otherwise specified, the operations of the present invention are all carried out at room temperature (20° C. to 25° C.).

[0030] Furthermore, in step 1), 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.

[0031] The DNase reagent used in the present invention can be purchased from Thermo Fisher Scientific (https: / / www.thermofisher.cn / order / catalog / product / EN0521).

[0032] In a third aspect, the present invention provides a PLGA nanosphere virus tracer having a protein capsid and a DNA marker prepared according to the method, wherein the particle size (x+2y) can be in the range of 65-105 nm, and can be controlled by the particle size x of the PLGA nanosphere containing the DNA marker according to the particle size of the target virus being traced, and fine-tuned by the thickness y of the protein capsid.

[0033] In a fourth aspect, the present invention provides the use of the tracer in the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied; wherein the pathogens include viruses.

[0034] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0035] (i) The PLGA nanosphere virus tracer provided by the present invention has a protein capsid and contains a DNA marker, which can effectively protect the encoding DNA and facilitate multi-source tracing and subsequent quantitative detection and analysis.

[0036] (ii) The particle size of the tracer is controlled by the size of the PLGA (DNA) core and the thickness of the protein capsid, so that it is close to the size of the target virus. This effectively ensures that the outermost capsid of the tracer is completely composed of selected proteins with properties similar to the isoelectric point and other properties of the target virus surface protein and a half-life greater than or equal to the half-life of the target virus, so that the surface properties of the tracer and its migration characteristics in the water and soil environment can be closer to the target virus.

[0037] (iii) By controlling the decay rate of the PLGA nanosphere virus tracer through the molecular weight of PLGA and different LA:GA (lactic acid:glycolic acid) ratios, a DNA virus tracer with a decay rate close to that of the target virus in the water and soil environment was obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Comparative diagram of protein-encapsulated DNA-encoded viral tracers of the prior art (a) and the present invention (b).

[0039] Figure 2 The figure is a schematic diagram of the production process of the PLGA nanosphere virus tracer of the present invention.

[0040] 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 PLGA nanosphere virus tracers in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] Experimental reagents: Vitamin EDa-tocopherol poly(ethylene glycol succinate) (TPGS), dimethyl sulfoxide (DMSO), dichloromethane (DCM, 99%), single-stranded DNA was obtained by annealing the single-stranded DNA in Table 1 and its reverse complementary strand, ultrapure water, tris (hydroxymethyl) aminomethane (Tris, 99.8%), sodium chloride (NaCl, 99.5%), MES buffer (pH 6.0), PBS buffer (pH 7.4). Quenching solution: 1 mg / mL casein solution. Storage buffer: 10 mg / mL glycine solution. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 10 mg / mL).

[0043] 75-85 kDa PLGA, poly(D,L-lactide-co-glycolide), was purchased from CD Bioparticles Drug Delivery (https: / / www.cd-bioparticles.net / polylactic-co-glycolic-acids?page=2). Human serum albumin was purchased from Sigma-Aldrich (https: / / www.sigmaaldrich.cn / CN / zh / substance / albuminfromh umanserum1234570024907). Deoxyribonuclease I (DNase I) was purchased from ThermoFisher Sci entific (https: / / www.thermofisher.cn / order / catalog / product / EN0521). DNase I was purchased from Thermo Fisher Scientific (https: / / www.thermofisher.cn / order / catalog / product / EN0521).

[0044] Table 1 DNA sequences (SEQ ID NO: 1-4)

[0045]

[0046] Note: The bold underline indicates the forward primer, and the italic underline indicates the reverse primer.

[0047] Example 1 PLGA nanosphere virus tracer with protein capsid and DNA marker

[0048] In this example, adenovirus whose nucleic acid is double-stranded DNA is used as the target virus to prepare PLGA nanosphere virus tracer.

[0049] 1. Preparation of the core of PLGA nanospheres containing DNA: 20 mg PLGA was mixed with 1 mL DMSO in a test tube and dissolved overnight. 5 μL 4×10 -6 The DNA solution of 100 μL was added to the PLGA / DMSO mixture and ultrasonicated on ice for 10 s (40% amplitude). The DNA / PLGA / DMSO mixture was added to a 50 mL beaker containing TPGS (20 mL 0.3% (w / v)) solution stirred on a magnetic stirrer using a syringe pump, with the syringe tip below the liquid surface of the solution. The emulsion was stirred in a fume hood for 4 hours, during which time the DMSO evaporated and the PLGA particles hardened to obtain the PLGA (DNA) nanosphere core ( Figure 2 a).

[0050] 2. The size and structure of the nanospheres 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 nanospheres was about 60 nm.

[0051] 3. To ensure that the DNA is completely encapsulated in the nanospheres and effectively protected, the PLGA (DNA) nanosphere cores were gently mixed in a 0.2U / μL DNase I solution and incubated at room temperature for 1 hour to digest the DNA fragments that were not completely encapsulated on the surface, and the PLGA (DNA) nanosphere cores with DNA completely protected were obtained ( Figure 2 b).

[0052] 4. Use EDC covalent coupling method to wrap protein in the outermost layer:

[0053] (1) Protein preparation: A human serum albumin solution was prepared in PBS buffer (pH 7.4) to obtain a protein solution with a concentration of 2 mg / mL.

[0054] (2) Covalent coupling of proteins: The PLGA (DNA) nanosphere solution was washed twice in MES buffer; after the second wash, the nanosphere precipitate was resuspended in 10 mL of MES buffer to obtain a PLGA (DNA) nanosphere solution with a concentration of 10 mg / mL; 10 mL of 10 mg / mL EDC was added dropwise while stirring at 18°C-25°C for 15 minutes; the solution was washed twice with PBS buffer and then resuspended in 5 mL of PBS buffer; 5 mL of protein solution was added thereto and reacted at 23°C for 3 hours with continuous stirring. After the reaction was completed, the solution was centrifuged and the supernatant was discarded. The nanosphere precipitate was resuspended in 10 mL of quenching solution. After stirring for 30 minutes, the solution was centrifuged again and the supernatant was discarded. The nanosphere precipitate was resuspended in storage buffer to obtain a PLGA nanosphere virus tracer, i.e., [Protein (PLGA (DNA))]( Figure 2 c), and stored at 4°C until use.

[0055] Selection of protein: The outermost layer is wrapped with a protein close to the isoelectric point of the adenovirus surface, and there are enough amino groups in the protein molecule to ensure that the protein molecule has enough amino sites to form covalent bonds with the carboxyl groups on the surface of the PLGA (DNA) nanospheres through dehydration condensation, and the half-life of the protein is greater than or equal to the half-life of the target virus. Human serum albumin is selected in this embodiment.

[0056] 5. The size and structure of the PLGA nanosphere virus tracer [Protein (PLGA (DNA))] were observed using a transmission electron microscope (TEM), and its zeta potential (ζ) and hydrodynamic diameter were determined using a Zetasizer Nano ZS. The PLGA nanosphere virus tracer [Protein (PLGA (DNA))] with a protein capsid and a DNA marker 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 c).

[0057] The half-life of the target virus adenovirus in surface water is 23 days. 85kDa PLGA with a half-life of about 22 days was selected to prepare PLGA(DNA). The half-life of human serum albumin is close to that of the core of PLGA(DNA). While simulating the surface characteristics of the virus, it will not affect the half-life of the DNA tracer.

[0058] 6. Quantitative detection of PLGA nanosphere virus tracers

[0059] The PLGA nanosphere virus tracer [Protein (PLGA (DNA))] 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 to simulate the protein digestion process to remove the outermost protein shell. PLGA was dissolved with DCM, and the released DNA was 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.

[0060] 7. The PLGA (DNA) nanospheres with uniform size and complete DNA protection obtained at the end of step 3 ( Figure 2 b) 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 the PLGA (DNA) nanospheres, the density of PLGA and the particle size of PLGA (DNA) measured in step 2. The number of encapsulated DNA copies is obtained by the detection method described in step 6. 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 PLGA (DNA) nanosphere on average.

[0061] Example 2 Application of PLGA Nanosphere Virus Tracer

[0062] Existing DNA virus tracers cannot accurately infer the migration and transformation process of the target virus from 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 and half-life in the soil close to those of the target virus, is used as a substitute for the target virus to trace 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 adenovirus.

[0063] 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 virus migration 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 each time only the conservative tracer KBr (used to reverse the soil hydrodynamic parameters) is put into the PLGA nanosphere virus tracer or target virus. 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, adsorption and desorption rates, and decay rates of the PLGA nanosphere virus tracer in the unsaturated zone soil and saturated zone soil under different conditions. The function of the filtration parameters, adsorption and desorption rates, and decay rates of the target virus is inferred.

[0064] 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 (PLGA nanosphere virus tracer, target virus, and KBr for reverse hydrodynamic parameters) were put into the flume together. The concentration curves of each tracer and target virus in the overlying water at different migration distances obtained in the experiment were combined with the convection-diffusion equation that considers the decomposition of pollutants and adsorption and desorption on particles, the sedimentation of suspended particles in water, and the scouring and siltation of water on bottom mud. The convection-diffusion parameters, adsorption-desorption rate, and decay rate of PLGA nanosphere virus tracers were used to reverse the convection-diffusion parameters, adsorption-desorption rate, and decay rate of the target virus under different irrigation periods and different ditch sections.

[0065] 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.

[0066] 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 PLGA nanosphere virus tracer of the present invention is encoded with different DNA sequences to mark the farmland and time of investment. Figure 3 The PLGA nanosphere virus tracers put into the four farmlands in the experiment encapsulated the four different DNA sequences in Table 1 respectively, and samples were collected from nearby groundwater wells and drainage ditches to detect the concentration changes of PLGA nanosphere virus tracers in groundwater and surface water. Then, the function of using PLGA nanosphere virus tracers to describe the migration and transformation process of the target virus obtained by the soil column and water tank experiments and simulations in the first two steps 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 migrating 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.

[0067] 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. PLGA nanosphere virus tracer, characterized in that: It is a PLGA nanosphere virus tracer with a protein capsid and a DNA marker, which consists of an inner core and a protein capsid wrapped in an outer shell; The inner core is a PLGA nanosphere containing a DNA marker, and a capsid with a thickness of y is formed on the outer side of the inner core by covalently coupling the protein through EDC; wherein 5 nm≤y≤20 nm; The particle size of the PLGA nanospheres containing DNA markers is x, 55 nm≤x≤65 nm; wherein the molecular weight of PLGA is 75-85 kDa, the molar ratio of LA:GA is 1:1, LA is lactic acid, and GA is glycolic acid; The particle size of the tracer is x+2y, 65 nm≤x+2y≤105 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 with properties close to those of the capsid protein of the target virus, and there are enough amino groups in the protein molecule to ensure that the protein molecule has enough amino sites to form covalent bonds with the carboxyl groups on the surface of the PLGA nanospheres through dehydration condensation, and the half-life of the protein is greater than or equal to the half-life of the target virus.

2. A method for preparing a PLGA nanosphere virus tracer, characterized in that: The following steps are involved: (1) After mixing PLGA, DMSO and DNA solution, the mixture was injected into TPGS solution by syringe pump, and the resulting emulsion was stirred in a fume hood for 4-8 hours to evaporate DMSO and harden PLGA particles, thereby preparing PLGA nanospheres containing DNA markers as the core, with a particle size of x, 55 nm ≤ x ≤ 65 nm; Among them, the molecular weight of PLGA is 75-85 kDa, the molar ratio of LA:GA is 1:1, LA is lactic acid, and GA is glycolic acid; (2) Enzymatically hydrolyze the DNA that is not completely wrapped on the outside of the core, wash and resuspend the core with MES buffer at pH 6.0 to obtain a PLGA (DNA) nanosphere solution with a concentration of 10 mg / mL; (3) Using EDC as a crosslinking agent, the carboxyl groups on the outside of the PLGA (DNA) nanospheres are brought into contact with the protein solution, and a protein capsid with a thickness of y is formed on the surface of the PLGA (DNA) nanospheres through covalent coupling with EDC; wherein 5 nm ≤ y ≤ 20 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 DNA solution: Prepare DNA solution with ultrapure water to obtain a concentration of 4×10 -6 M DNA solution; 2) Preparation of DNA-labeled PLGA nanospheres: 20 mg of PLGA was mixed with 1 mL of DMSO and dissolved overnight to obtain a PLGA / DMSO mixture; 5 μL of the prepared DNA solution was added to the PLGA / DMSO mixture and sonicated on ice for 10 s to obtain a DNA / PLGA / DMSO mixture; the DNA / PLGA / DMSO mixture was injected into 20 mL of 0.3 w / v% TPGS solution using a syringe pump while stirring on a magnetic stirrer, and the resulting emulsion was stirred in a fume hood for 4 to 8 hours to evaporate the DMSO and harden the PLGA particles, thereby obtaining a solution of hardened PLGA particles; 3) Enzymatic hydrolysis of DNA that is not completely wrapped outside the PLGA nanospheres containing DNA markers: contacting the solution of hardened PLGA particles with a DNA enzyme reagent to enzymatically hydrolyze the DNA exposed outside the nanospheres to obtain a PLGA nanosphere solution containing DNA markers, i.e., a PLGA (DNA) nanosphere solution; 4) Preparation of protein solution: Prepare the protein solution with PBS buffer to obtain a protein solution with a concentration of 2 mg / mL; 5) Covalent coupling of proteins: The PLGA (DNA) nanosphere solution was washed twice in MES buffer; after the second wash, the nanosphere precipitate was resuspended in 10 mL of MES buffer to obtain a PLGA (DNA) nanosphere solution with a concentration of 10 mg / mL; 10 mL of 10 mg / mL EDC was added dropwise while stirring at 18°C-25°C for 15 minutes; the nanospheres were washed twice with PBS buffer and then resuspended with 5 mL of PBS buffer; 1.85-18.5 mL of the protein solution was added thereto, and the mixture was reacted at 18°C-25°C for 2-4 hours with continuous stirring. After the reaction was completed, the supernatant was discarded by centrifugation, and the nanosphere precipitate was resuspended in 10 mL of quenching solution. After stirring for 30 minutes, the supernatant was discarded by centrifugation again, and the nanosphere precipitate was resuspended in storage buffer to obtain a PLGA nanosphere virus tracer [Protein (PLGA (DNA))], i.e., a PLGA nanosphere virus tracer with a protein capsid and a DNA marker; Wherein, the pH of the MES buffer is 6.0, and the pH of the PBS buffer is 7.4; The quenching solution is a 1 mg / mL casein solution; The storage buffer was a 10 mg / mL glycine solution.

4. The method according to claim 3, characterized in that In step 1), 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.

5. A PLGA nanosphere virus tracer having a protein capsid and a DNA marker, prepared according to the method of any one of claims 2 to 4.

6. The tracer according to claim 5, characterized in that Its particle size is x+2y, 65 nm≤x+2y≤105 nm.

7. Use of the tracer according to claim 1, 5 or 6 in the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied; in, The pathogens include viruses.

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