A fusion protein BVP10-GS-R5 and nano-silicon dioxide acaricide and its preparation method and application

Abstract

The present invention provides a fusion protein BVP10‑GS‑R5 and a nano-silicon dioxide acaricide and a preparation method and application thereof, belonging to the technical field of biological pesticides. The amino acid sequence of the fusion protein BVP10‑GS‑R5 of the present invention is shown in SEQ ID NO.2. The present invention utilizes R5 peptide and acaricide protein BVP10 to construct a fusion protein, and then uses the fusion protein to prepare a nano-silicon dioxide acaricide, which can react at room temperature and pressure, with mild and controllable conditions, good biocompatibility and environmental benefits, and solves the problem of harsh preparation conditions of nano-silicon dioxide preparations in the prior art. And compared with the fusion protein BVP10‑GS‑R5, the nano-silicon dioxide acaricide prepared by the present invention has better sustained release, adhesion and acaricide activity, and is suitable for large-scale promotion and application.

Description

Technical Field

[0001] The invention relates to the technical field of biological pesticides, and in particular to a fusion protein BVP10-GS-R5 and a nano-silicon dioxide acaricide, and a preparation method and application thereof. Background Art

[0002] Mites belong to the class Arachnida of the phylum Arthropoda. Most common harmful mites belong to the orders Acari and Acarina, and are one of the important pests that harm a variety of crops. The main phytophagous mites include spider mites, panonychus mites, gall mites, rust mites, etc., which harm vegetables, fruit trees, cotton and other crops, often causing devastating disasters. Due to their basic ecological countermeasures such as short reproduction cycle, small size, fast development, strong reproductive capacity and easy to develop drug resistance, phytophagous mites have become one of the pests that are difficult to control. Long-term chemical control has made the drug resistance of harmful mites increasingly aggravated, resulting in large amounts of drugs used for pest mite control, high costs, poor effects, and serious environmental pollution. With the increasing attention to environmental issues and the requirements for green and high-quality development of agriculture, it is urgent to develop new miticides that are safe, efficient, long-lasting, environmentally friendly and not prone to resistance.

[0003] BVP10 is a small molecule protein with acaricidal activity found in the supernatant of the fermentation broth of Bacillus vallismortis NBIF-001. BVP10 recombinantly expressed in Escherichia coli by genetic engineering technology showed good acaricidal activity in indoor experiments. Compared with chemical synthetic agents, protein substances are easily affected by physical and chemical factors such as temperature and pH, which limits their application in open environments. Nanopesticide technology can improve the activity and adhesion of pesticide active ingredients. Nanosilica has the advantages of small particle size and large specific surface area, and is one of the more ideal nanopesticide preparation materials. However, the preparation conditions of nanosilica preparations in the prior art are harsh and are not suitable for the preparation of nanosilica acaricides with protein as active substances. Therefore, it is urgent to develop a method for preparing nanosilica acaricides with mild reaction conditions. Summary of the invention

[0004] The purpose of the present invention is to provide a fusion protein BVP10-GS-R5 and a nano-silica acaricide and a preparation method and application thereof. When the recombinantly expressed R5 peptide is mixed with silicic acid at room temperature and pressure, the amino group in the R5 peptide interacts with the silicic acid monomer to initiate the formation of siloxane bonds, thereby mediating the biological deposition of nano-silica in a short time. The reaction can be carried out at room temperature and pressure, the conditions are mild and controllable, and it has good biocompatibility and environmental benefits, which solves the technical problem that the reaction conditions of nano-silica preparations in the prior art are harsh and not suitable for the preparation of nano-silica acaricides with proteins as active substances.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a fusion protein BVP10-GS-R5, the nucleic acid sequence encoding the fusion protein BVP10-GS-R5 is shown as SEQ ID NO.1, and the amino acid sequence of the fusion protein BVP10-GS-R5 is shown as SEQ ID NO.2.

[0007] The present invention also provides a recombinant strain expressing the fusion protein BVP10-GS-R5, wherein the recombinant strain is Escherichia coli BLR (DE3).

[0008] The present invention also provides a method for preparing a nano-silicon dioxide acaricide, comprising the following steps:

[0009] S1. Dissolve the fusion protein BVP10-GS-R5 in phosphate buffer to obtain a fusion protein suspension;

[0010] S2. mixing methyl silicate with HCl solution and hydrolyzing to obtain a silicic acid mother liquor;

[0011] S3. The fusion protein suspension is mixed with the silicic acid mother solution, reacted at 28-32° C. for 3.5-4.5 hours, and then centrifuged. The resulting precipitate is the nano-silicon dioxide acaricide.

[0012] Preferably, the concentration of the phosphate buffer is 48-52 mM.

[0013] Preferably, the concentration of the fusion protein BVP10-GS-R5 in the fusion protein suspension is 280-320 μg / mL.

[0014] Preferably, the volume ratio of the molar amount of methyl silicate to the HCl solution is 0.5 mmol: 0.8-1 mL; and the concentration of the HCl solution is 0.8-1.2 mM.

[0015] Preferably, the hydrolysis time is 8 to 12 minutes.

[0016] Preferably, the volume ratio of the silicic acid mother solution to the fusion protein suspension is 1:8-10.

[0017] The invention also provides a nano silicon dioxide acaricide prepared by the preparation method.

[0018] The present invention also provides the use of the nano silicon dioxide acaricide in preventing and controlling mites or preparing a medicine for preventing and controlling mites.

[0019] By adopting the above technical scheme, the present invention has the following beneficial effects: the amino acid sequence of the fusion protein BVP10-GS-R5 of the present invention is shown in SEQ ID NO.2. The present invention uses R5 peptide and acaricidal protein BVP10 to construct a fusion protein, and then uses the fusion protein to prepare a nano-silicon dioxide acaricide, which can react at room temperature and pressure, with mild and controllable conditions, good biocompatibility and environmental benefits, and solves the problem of harsh preparation conditions of nano-silicon dioxide preparations in the prior art. And the nano-silicon dioxide acaricide prepared by the present invention has better sustained release, adhesion and acaricidal activity than the fusion protein BVP10-GS-R5, and is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Band 1 is the SDS-PAGE band diagram of the ELP tag-purified BVP10-GS-R5 fusion protein, and Band 2 is the SDS-PAGE band diagram of the supernatant of E. coli cell lysate. Figure 2 This is the band of the ELP tag fusion protein purified by the reversible phase change cycle method. Figure 3 This is the band of BVP10-GSx1-R5 after the tag was removed.

[0021] Figure 2 Fourier transform infrared spectra of silicon dioxide (a) and nano-silica acaricide (b).

[0022] Figure 3 This is a scanning electron microscope (SEM) image of nano-silica acaricide.

[0023] Figure 4 This is the sustained release curve of the fusion protein BVP10-GS-R5 in the nano-silica acaricide.

[0024] Figure 5 Fluorescence images of deposits on the leaf surface before and after cleaning the leaf with deionized water. DETAILED DESCRIPTION

[0025] The present invention provides a fusion protein BVP10-GS-R5, the nucleic acid sequence encoding the fusion protein BVP10-GS-R5 is shown as SEQ ID NO.1, and the amino acid sequence of the fusion protein BVP10-GS-R5 is shown as SEQ ID NO.2.

[0026] The present invention also provides a recombinant strain expressing the fusion protein BVP10-GS-R5, wherein the recombinant strain is Escherichia coli BLR (DE3).

[0027] The present invention also provides a method for preparing a nano-silicon dioxide acaricide, comprising the following steps:

[0028] S1. Dissolve the fusion protein BVP10-GS-R5 in phosphate buffer to obtain a fusion protein suspension;

[0029] S2. mixing methyl silicate with HCl solution and hydrolyzing to obtain a silicic acid mother liquor;

[0030] S3. The fusion protein suspension is mixed with the silicic acid mother solution, reacted at 28-32° C. for 3.5-4.5 hours, and then centrifuged. The resulting precipitate is the nano-silicon dioxide acaricide.

[0031] The present invention first obtains a gene fragment expressing the fusion protein BVP10-GS-R5 shown in SEQ ID NO: 2 by artificial synthesis, and the nucleic acid sequence of the gene fragment is shown in SEQ ID NO.1.

[0032] SEQ ID NO.1:

[0033] TTAAAAAGTAAAATTAAAAAAATAGCCGGTGCGGCTGTAATTGC

[0034] TGGAGCATTATTGGTTTCTGTGTCGCCAGCAAAAGCAGAAACTCTTCC

[0035] GGGATTGATCTGGGTCATGGACAAATATACTGACGCTTTTAAATTCGAC

[0036] ATGTTTATTAAAAACGGAAATGAAAAATGGGTTTATGTTTACACAAGAT

[0037] TATCCGATGGCAAAGTTGATGTGGAAGAAGTAAAGTGTGAAACAATTT

[0038] ATAAAGATCGATGCTATGTAAAGCAGGGCGGTGGTGGTAGCTCTTCCA

[0039] AGAAATCTGGTAGCTACTCTGGTTCTAAAGGTTCCAAACGTCGTATCTTG.

[0040] SEQ ID NO.2:

[0041] LKSKIKKIAGAAVIAGALLVSVSPAKAETLPGLIWVMDKYTDAFKF

[0042] DMFIKNGNEKWVYVYTRLSDGKVDVEEVKCETIYKDRCYVKQGGGGSS SKKSGSYSGSKGSKRRIL.

[0043] The R5 peptide is a repeating unit sequence found in the cell wall protein silaffin of the diatom Cylindrotheca spinosa. The amino acid sequence of the R5 peptide is shown in SEQ ID NO:3.

[0044] SEQ ID NO: 3: SSKKSGSYSGSKGSKRRIL.

[0045] The amino acid sequence of BVP10 protein is shown in SEQ ID NO:4.

[0046] SEQ ID NO: 4:

[0047] LKSKIKKIAGAAVIAGALLVSVSPAKAETLPGLIWVMDKYTDAFKF DMFIKNGNEKWVYVYTRLSDGKVDVEEVKCETIYKDRCYVKQ.

[0048] Then the BVP10-GS-R5 gene sequence is inserted into the expression vector pET24a(+)-ELP, which carries an elastin-like polypeptide (ELP) purification tag. The BVP10-GS-R5 gene sequence is connected to the ELP tag via a thrombin cleavage site (LVPRGS) to obtain a recombinant plasmid pET24a(+)-ELP-BVP10-GS-R5. The recombinant plasmid is transformed into an Escherichia coli BLR(DE3) host cell to obtain an expression strain BLR(DE3) / pET24a(+)-ELP-BVP10-GS-R5 carrying the BVP10-GS-R5 gene.

[0049] A single clone of the recombinant bacteria was picked and inoculated into a TB liquid medium, and kanamycin was added for shaking culture; the concentration of kanamycin in the TB liquid medium was 90-110 ng / μL, more preferably 95-105 ng / μL, and more preferably 100 ng / μL. The shaking culture temperature was 35-39°C, more preferably 36-38°C, and more preferably 37°C; the shaking culture speed was 180-220 rpm, more preferably 190-210 rpm, and more preferably 200 rpm. The shaking culture was continued until OD 600Reach 0.8, add isopropyl-BD thiogalactoside (IPTG) for induction culture, so that the final concentration of IPTG is 0.18-0.22mmol / L, more preferably 0.19-0.21mmol / L, more preferably 0.2mmol / L; the temperature of the induction culture is 15-17°C, more preferably 15.5-16.5°C, more preferably 16°C; the time of the induction culture is 45-50h, more preferably 46-49h, more preferably 48h. After the induction culture is completed, the IPTG induced culture is collected, the bacteria are collected by centrifugation, the bacteria are resuspended and then the bacteria are broken by an ultrasonic cell disruptor, and the bacterial lysate supernatant is collected by centrifugation. Then, impurities are removed by filtration with a 0.22μm pore size filter membrane, and the fusion protein ELP-BVP10-GS-R5 is purified by the reversible phase change cycle (ITC) technology of the ELP tag, and then the purified target protein BVP10-GS-R5 is obtained by removing the ELP tag with thrombin.

[0050] In the present invention, the fusion protein BVP10-GS-R5 is dissolved in a phosphate buffer to a final concentration of 280-320 μg / mL, more preferably 290-310 μg / mL, and more preferably 300 μg / mL; the concentration of the phosphate buffer is 48-52 mM, more preferably 49-51 mM, and more preferably 50 mM. Then, methyl silicate is mixed with an HCl solution for hydrolysis, and the concentration of the HCl solution is 0.8-1.2 mM, more preferably 0.9-1.1 mM, and more preferably 1 mM; the molar amount and volume ratio of methyl silicate to hydrochloric acid solution is 0.5 mmol: 0.8-1 mL, more preferably 0.5 mmol: 0.85-0.95 mL, and more preferably 0.5 mmol: 0.926 mL; the hydrolysis time is 8-12 min, more preferably 9-11 min, and more preferably 10 min. Then, the silicic acid mother solution is taken and added to the fusion protein suspension for reaction, and then centrifuged, the precipitate is collected, and washed to obtain the nano-silicon dioxide acaricide. The volume ratio of the silicic acid mother solution to the fusion protein suspension is 1:8-10, more preferably 1:8.5-9.5, and more preferably 1:9. The reaction temperature is 28-32°C, more preferably 29-31°C, and more preferably 30°C. The reaction time is 3.5-4.5h, and more preferably 4h.

[0051] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] Example 1. Preparation of nano-silicon dioxide acaricide

[0053] 1. Preparation of Fusion Protein

[0054] The BVP10-GS-R5 gene fragment shown in SEQ ID NO: 1 was obtained by artificial synthesis (commissioned by Jiangsu GenScript Biotech Co., Ltd. for synthesis), and then the fragment was inserted into the E. coli expression vector pET24a(+)-ELP using the NovoRec plus One step PCR Cloning Kit (Suzhou Nearshore Protein Technology Co., Ltd.) according to the instructions. The BVP10-GS-R5 gene sequence was connected to the ELP tag through the thrombin cleavage site (LVPRGS) to obtain the recombinant plasmid pET24a(+)-ELP-BVP10-GS-R5. The recombinant plasmid was mixed with E. coli competent cells BLR (DE3) (Shanghai Weidi Biotechnology Co., Ltd.), left on ice for 25 minutes, then heat-shocked in a 42°C water bath for 45 seconds, and 700 μL of sterile LB medium without antibiotics was added. After mixing, the mixture was revived at 37°C, 200 rpm for 60 minutes, and then spread on a plate and cultured in a 37°C incubator overnight. A single clone was picked to obtain the expression strain BLR(DE3) / pET24a(+)-ELP-BVP10-GS-R5 carrying the BVP10-GS-R5 gene.

[0055] The recombinant bacterial monoclonal was inoculated into 5 mL of LB medium for overnight culture. Then the bacterial solution was inoculated into TB liquid medium, kanamycin was added to a final concentration of 100 ng / μL, and cultured at 37°C, 200 rpm / min until the OD 600 When the pH value reaches 0.8, add isopropyl-BD thiogalactoside (IPTG) with a final concentration of 0.2mmol / L and induce culture at 16℃ for 48h. Collect IPTG-induced culture, collect bacteria by centrifugation at 4℃, 5000g for 5min, resuspend the bacteria and use ultrasonic cell disruptor to break the bacteria, and collect the supernatant of bacterial lysate by centrifugation at 4℃, 14000g for 30min. Use 0.22μm pore size filter to remove impurities, and use the reversible phase change cycle (ITC) technology of ELP tag to purify the fusion protein ELP-BVP10-GS-R5. The specific operation is: add NaCl with a final concentration of 2.5M to the supernatant of bacterial lysate, incubate at 30℃ for 10min, then centrifuge at room temperature for 10min at 14000g, resuspend the precipitate with pre-cooled PBS, repeatedly blow to dissolve the precipitate, then centrifuge at 4℃, 14000g for 10min, and discard the precipitate. The above cycle was repeated once more to obtain a fusion protein with an ELP tag. Thrombin was then used to cleave the ELP tag at 37°C for 24 hours to obtain the purified target protein BVP10-GS-R5 (protein sequence shown in SEQ ID NO.2, theoretical molecular weight of about 12.4 kDa). The purified recombinant protein was subjected to SDS-PAGE electrophoresis detection. The electrophoresis results were as follows: Figure 1As shown, the target band is substantially consistent with the expected molecular weight of the recombinant protein, which proves that the BVP10-GS-R5 fusion protein of the present invention is successfully expressed in Escherichia coli BLR (DE3).

[0056] 2. Preparation of Nano-Silica Acaricide

[0057] The nano-silica acaricide was prepared using the fusion protein BVP10-GS-R5 as a template and methyl silicate as a silicon source. The specific operation was as follows: the fusion protein BVP10-GS-R5 was dissolved in 50mM phosphate buffer to a final concentration of 300μg / mL. 0.5mmol methyl silicate was hydrolyzed in 0.926mL 1mM HCl solution for 10min to prepare a silicic acid mother solution. 1μL of the silicic acid mother solution was added to 9μL of the BVP10-GS-R5 fusion protein suspension, and the mixture was allowed to stand at 30°C for 4h, then centrifuged at room temperature for 2min at 14000g to discard the supernatant, and the precipitate was collected and washed with deionized water to obtain the nano-silica acaricide.

[0058] Example 2. Determination of properties of nano-silicon dioxide

[0059] 1. Fourier transform infrared spectroscopy (FTIR) detection of nano-silica acaricide

[0060] The characteristic peaks of the nano-silicon dioxide acaricide prepared in Example 1 were detected by FTIR. Figure 2 As shown in the spectrum, there is a Si-O-Si asymmetric stretching peak (1101cm -1 ) and Si-O symmetric stretching peak (802cm -1 ), proving the synthesis of nano-silicon dioxide. -1 and 1534cm -1 The two peaks at correspond to the C=O stretching vibration of the amide I band of the polypeptide backbone and the NH bending vibration of the amide II band, respectively, indicating that the protein is fixed into the nano-silica.

[0061] 2. Scanning electron microscopy (SEM) characterization of the microstructure of nano-silica acaricide

[0062] The nano-silicon dioxide prepared in Example 1 was washed and dried, then dispersed and fixed on a carbon conductive double-sided tape, and gold was sprayed on the surface for 60 seconds, and then the microstructure of the nano-silicon dioxide was observed using a SEM. Figure 3 It can be seen that the smaller nano-silicon dioxide particles aggregate to form large particles with a diameter of about 500nm, and there are many gaps between the particles.

[0063] 3. Determination of drug loading capacity of nano-silica acaricide

[0064] The protein concentration of nano-silica before and after synthesis was determined using a Bradford protein concentration assay kit (Beijing Coolbo Technology Co., Ltd.). The mass of silica was quantitatively detected by molybdate colorimetry. The protein encapsulation rate of the nano-silica acaricide was calculated to be 91.5% and the drug loading was 8.1%.

[0065] The calculation formulas for encapsulation efficiency (EE) and drug loading content (LC) are:

[0066]

[0067] Among them, m 1 Refers to the total amount of protein added to the reaction system, m 2 Refers to the amount of free protein in the supernatant after the reaction, m 0 Refers to the mass of silica produced by the reaction.

[0068] 4. Slow-release properties of nano-silica acaricide

[0069] A certain amount of nano-silica acaricide was mixed with PBS and then released by oscillation at 37°C. One day after release, the supernatant was taken to determine the protein concentration using a Bradford kit, and the same volume of fresh PBS was added. Then, samples were taken every 3 days in the same way. The sustained release curve was drawn according to the cumulative release formula, and the release behavior of the protein was kinetically fitted using the Ritger-peppas model, and the fitting equation Q = 2.7259t 0.7766 , R 2 =0.9894. Figure 4 It can be seen that the protein in the nano-silica carrier is slowly released over time, and the cumulative release rate reaches 30% on day 21. In the Ritger-Peppas equation, 0.45≤n=0.7766≤0.89, indicating that the release mechanism is non-Fick diffusion, and is released through the combined action of diffusion and skeleton dissolution.

[0070] The cumulative release rate (Q) is calculated as:

[0071]

[0072] Wherein, W is the mass of the protein loaded in the nanoparticles; V0 is the total volume of the sustained-release medium; Ve is the volume of each sampling; Ci is the protein concentration of the sustained-release solution of the i-th sampling (mg / mL); and n is the number of samplings.

[0073] 5. Acaricidal activity of nano-silica acaricide

[0074] Protein BVP10, fusion protein BVP10-GS-R5 and nano-silica acaricide were mixed with PBS buffer to obtain suspensions with a gradient of 30 μg / mL, 15 μg / mL, 7.5 μg / mL and 3.75 μg / mL of protein concentration, which were slowly released in a shaker at 37°C and 200 rpm for 14 days. The spider mite killing activity was determined using the spider mite slide immersion method. Double-sided tape was cut into squares of about 2 cm and attached to one end of the slide. The backs of female adult mites with uniform size, bright body color and active movements were attached to the double-sided tape. After being placed in a biochemical incubator at a temperature of 25°C and a relative humidity of 85% for 2 to 3 hours, dead, injured or inactive individuals were removed by microscopic examination. One end of the glass slide with mites was immersed in these different concentrations of nano-silica acaricide and protein suspension, and gently shaken for 5 seconds before being taken out. The excess liquid around the mite body and the surrounding area was quickly sucked off, and the glass slide was placed in a biochemical incubator for 24 hours. PBS buffer was used as the control group. The test results were observed with binoculars, and the mite body was touched to determine whether the mite was dead. The mite was considered dead if its feet did not move. Each group was repeated 3 times.

[0075] According to the above experimental steps, the bioassay results of the acaricide protein BVP10, the fusion protein BVP10-GS-R5 and the nano-silicon dioxide acaricide on the two-spotted spider mite (Tetranychus urticae) are shown in Table 1.

[0076] Table 1 Experimental data on the determination of the acaricidal activity of Tetranychus urticae

[0077]

[0078]

[0079] SPSS19.0 data processing software was used to calculate the LC of the acaricidal protein BVP10 against two-spotted spider mites. 50 The LC of fusion protein BVP10-GS-R5 against Tetranychus urticae was 22.18 μg / mL. 50 The LC value of nano-silica acaricide against Tetranychus urticae was 7.61 μg / mL, indicating that the acaricidal activity was improved after protein fusion expression. 50 It is 3.11 μg / mL, and the synergistic ratio (SR) relative to the fusion protein BVP10-GS-R5 is 2.45, indicating that the nano-silica acaricide has a significant synergistic acaricidal effect.

[0080] 6. Adhesion of Nano-Silica Acaricide

[0081] Dissolve fluorescein isothiocyanate (FITC) in dimethyl sulfoxide (DMSO) to prepare a 5 mg / mL solution. Add 10 μL of FITC solution to 1 mL of the aqueous suspension of the fusion protein BVP10-GS-R5 and the nano-silica acaricide, incubate at room temperature in the dark for 3 hours, and then dialyze in ultrapure water at low temperature in the dark for 24 hours. Add the dialyzed suspensions dropwise onto broad bean leaves and air-dry naturally at room temperature. Use a stereofluorescence microscope (Leica M165FC, Germany) to collect fluorescent images of the sediments on the leaf surface. Then, wash the leaves once with deionized water, air-dry at room temperature, collect fluorescent images, wash the leaves 10 times with deionized water, air-dry at room temperature, collect fluorescent images, and the results are as follows: Figure 5 As shown. Figure 5 It can be seen that the protein BVP10-GS-R suspension was completely washed away after one wash, while the fluorescence area of ​​the nano-silica acaricide did not change significantly after 10 washes. The fluorescence area of ​​the leaf surface deposits was calculated using ImageJ software, and the fluorescence area before washing was set to 100%, and the residual rate of the fluorescent deposits after washing was calculated. It was calculated that the residual rates of the nano-silica acaricide were 99.30% and 90.50% after washing once and 10 times, respectively, indicating that the nano-silica acaricide has better adhesion than the fusion protein BVP10-GS-R5.

[0082] From the above examples, it can be seen that the present invention provides a fusion protein BVP10-GS-R5 and a nano-silicon dioxide acaricide and a preparation method and application thereof. The nano-silicon dioxide acaricide of the present invention has good sustained release, adhesion and acaricide activity, and is suitable for large-scale promotion and application.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a nano-silicon dioxide acaricide, characterized in that: The following steps are involved: S1. Dissolve the fusion protein BVP10-GS-R5 in a phosphate buffer to obtain a fusion protein suspension; the concentration of the phosphate buffer is 48 to 52 mM, and the concentration of the fusion protein BVP10-GS-R5 in the fusion protein suspension is 280 to 320 µg / mL; S2. Mixing methyl silicate with HCl solution and hydrolyzing to obtain a silicic acid mother solution; the volume ratio of methyl silicate to HCl solution is 0.5mmol:0.8-1mL; the concentration of the HCl solution is 0.8-1.2mM, and the hydrolysis time is 8-12min; S3. The fusion protein suspension is mixed with the silica mother solution, reacted at 28-32° C. for 3.5-4.5 h and then centrifuged to obtain a precipitate which is a nano-silica acaricide; the volume ratio of the silica mother solution to the fusion protein suspension is 1:8-10; The nucleotide sequence encoding the fusion protein BVP10-GS-R5 is shown in SEQ ID NO.1, and the amino acid sequence of the fusion protein BVP10-GS-R5 is shown in SEQ ID NO.

2.

2. The nano-silicon dioxide acaricide prepared by the preparation method according to claim 1.

3. Use of the nano-silicon dioxide acaricide according to claim 2 in controlling crop mites or preparing pesticides for controlling mites.

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