Novel biological nano anti-mosquito drug based on iron-sulfur cluster synthetic protein as target spot

Abstract

The invention discloses a novel biological nano anti-mosquito drug based on iron-sulfur cluster synthetic protein as a target spot, belongs to the technical field of biological drugs, and develops a chitosan-sodium tripolyphosphate-dsNFS1 (CS-TPP-dsNFS1) biological nano anti-mosquito technology by taking AalbNFS1 as a target spot. A fluorescent quantitative PCR (polymerase chain reaction) method is used for detecting the expression level of tissue AalbNFS1 mRNA (messenger ribonucleic acid) at different time points after eclosion of aedes albopictus larvae fed with the CS-TPP-dsNFS1 nanoparticles. In addition, three days and four days after eclosion, the aedes albopictus fed with the CS-TPP-dsNFS1 nanoparticles are subjected to flight capability and blood sucking capability detection respectively, and then reproductive capability detection is performed. The result shows that the CS-TPP-dsNFS1 obviously inhibits the flying, blood sucking and reproductive capacities of aedes albopictus, which indicates that the CS-TPP-dsNFS1 taking the AalbNFS1 as the target spot is a novel developable anti-mosquito drug.

Description

Technical Field

[0001] The invention belongs to the technical field of biological medicines, and in particular relates to a novel biological nano mosquito repellent based on iron-sulfur cluster synthetic protein as a target. Background Art

[0002] Mosquitoes are the vectors of many mosquito-borne disease viruses. To date, these mosquito-borne diseases have caused many major public health incidents worldwide. Since most mosquito-borne disease viruses lack effective vaccines, people mainly adopt various mosquito prevention and control measures to deal with the threats and pressures that mosquito-borne diseases bring to the public health system. Since insecticides may pollute the environment and threaten human health, and more and more mosquito species have begun to develop resistance to various insecticides, researchers have begun to explore other mosquito prevention strategies. In recent years, mosquito prevention strategies based on genetic methods have attracted more and more attention due to their species specificity and low toxicity. RNA interference based on double-stranded RNA (dsRNA) is a gene silencing strategy that has been successfully applied to insects. However, the successful application of dsRNA in practical mosquito prevention strategies must solve three major problems: (1) How to efficiently and cost-effectively deliver these nucleotides to mosquitoes? (2) How to maintain the stability of these nucleotides in the environment and in mosquitoes? (3) How can dsRNA be used to simultaneously inhibit the flight, blood-sucking and reproductive ability of mosquitoes? Studies have shown that chitosan-sodium tripolyphosphate nanosystem can be used as a low-cost delivery system to effectively improve the stability of dsRNA in the environment and in mosquitoes, thereby solving the first two problems. However, how to simultaneously inhibit the flight, blood-sucking and reproductive ability of mosquitoes has always been an unresolved problem.

[0003] Iron-sulfur clusters play an important role in cellular respiration and metabolism. In mammals, impaired iron-sulfur cluster synthesis can inhibit cellular respiration and thus muscle function. Since the flight and blood-sucking behaviors of mosquitoes are dependent on muscle function, disrupting the biosynthesis of iron-sulfur clusters in mosquito muscle tissue can simultaneously inhibit its flight and blood-sucking abilities, and the reduction in blood-sucking ability can further inhibit the reproductive ability of mosquitoes. Therefore, the biosynthesis of iron-sulfur clusters in mosquito muscle tissue is an ideal target for mosquito control. However, since the mechanism of iron-sulfur cluster biosynthesis in mosquitoes has been unknown, this target has not been utilized. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a new type of bio-nano mosquito repellent based on iron-sulfur cluster synthetic protein as a target.

[0005] A control drug for Aedes albopictus is a dsRNA with the iron-sulfur cluster synthesis core protein AalbNFS1 as a target; the nucleic acid sequence of the AalbNFS1 is shown in the sequence table SEQ ID NO: 1.

[0006] The nucleotide sequence of the dsRNA is as shown in SEQ ID NO: 2;

[0007] dsNFS1, constructed into chitosan-sodium tripolyphosphate-dsNFS1 bionanomedicine;

[0008] The chitosan-sodium tripolyphosphate-dsNFS1 is prepared by the following method:

[0009] 1) Prepare sodium tripolyphosphate TPP solution, and adjust the pH value to 4.0-5.0;

[0010] 2) TPP solution is coupled with CS and dsNFS1;

[0011] 3) Centrifuge, take the precipitate and disperse;

[0012] Step 3) The coupling is carried out by sequentially adding dsNFS1 and chitosan solution to the TPP solution under magnetic stirring at 600 rpm for reaction;

[0013] The weight ratio of chitosan, sodium tripolyphosphate and dsNFS1 in the bionanomedicine is 5:1:0.0064;

[0014] The centrifugation was carried out at 12000 g for 5 minutes at 4°C. The obtained precipitate was washed three times with deionized water, dispersed in deionized water, and stored in a refrigerator at 4°C.

[0015] The reaction time is 60 minutes.

[0016] The present invention provides a novel bio-nano control drug for Aedes albopictus, especially the identification of the core protein AalbNFS1 of the iron-sulfur cluster synthesis of Aedes albopictus, which is the first identification; and for the first time, the iron-sulfur cluster synthesis protein AalbNFS1 is used as a target to control Aedes albopictus by constructing a bio-nano system. The results show that the chitosan-sodium tripolyphosphate-dsNFS1 bio-nano drug targeting AalbNFS1 can significantly inhibit the flight, blood-sucking and reproductive behavior of Aedes albopictus, thereby achieving a good control effect of Aedes albopictus.

[0017] The present invention identifies the iron-sulfur cluster synthesis core protein AalbNFS1 of Aedes albopictus for the first time, studies the biological function of this protein in vivo, and assembles the dsRNA targeting the AalbNFS1 gene (named dsNFS1) into the chitosan-sodium tripolyphosphate nanosystem for the first time to form the chitosan-sodium tripolyphosphate-dsNFS1 (hereinafter referred to as CS-TPP-dsNFS1) bio-nanosystem. CS-TPP-dsNFS1 was used to feed the larvae of Aedes albopictus, and its mosquito repellent effect was investigated in the adult stage. The results show that the CS-TPP-dsNFS1 bio-nanosystem can significantly inhibit the flight, blood-sucking and reproductive behaviors of Aedes albopictus. The identified iron-sulfur cluster synthesis core protein AalbNFS1 of Aedes albopictus is used as a dsRNA target, and the dsNFS1 targeting the AalbNFS1 gene is assembled into the CS-TPP-dsNFS1 bio-nanosystem for use as a mosquito repellent, which is a new drug development for the prevention and control of Aedes albopictus. Compared with existing mosquito control strategies, the CS-TPP-dsNFS1 bio-nanosystem has the following advantages: (1) Compared with existing mosquito control methods based on genetic technology, this method does not require the construction of a mosquito population with genetic defects, nor does it need to worry about the reproductive disadvantages of a mosquito population with genetic defects. Simply spreading the CS-TPP-dsNFS1 bio-nanosystem into the water can achieve the mosquito control effect of simultaneously inhibiting the flight, blood-sucking and reproductive behavior of Aedes albopictus, saving time and effort and having low costs. (2) Compared with insecticides, this method has the advantages of high species specificity, low toxicity, no pollution, no drug resistance, and low cost. (3) Compared with mosquito lamps, this method does not require electricity supply and has a wider coverage range of effects, with better universality and ease of operation. The following is the research process of the development of this new mosquito repellent: BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of similarity and conserved motif analysis of AalbNFS1;

[0019] Figure 2 The relative expression levels of AalbNFS1 mRNA in the Unloaded, CS-TPP, dsNFS1, and CS-TPP-dsNFS1 groups from the first to the sixth day after the emergence of Aedes albopictus. The same lowercase letters in the bar graph indicate no significant difference, while different lowercase letters indicate significant difference (P<0.05, one-way ANOVA);

[0020] Figure 3 This is a schematic diagram of the experiment to test the flight ability of Aedes albopictus;

[0021] Figure 4The experimental results of (A) flight time and (B) flight frequency of Aedes albopictus on the third day after emergence in the Unloaded, CS-TPP, dsNFS1, and CS-TPP-dsNFS1 groups. ns means no significant difference, and the P value is calculated by unpaired t-test;

[0022] Figure 5 This is a schematic diagram of the experiment to test the blood-sucking ability of Aedes albopictus;

[0023] Figure 6 The experimental results of (A) blood-sucking response time, (B) blood-sucking frequency, and (C) blood-sucking volume of Aedes albopictus in the Unloaded, CS-TPP, dsNFS1, and CS-TPP-dsNFS1 groups on the fourth day after emergence. ns means no significant difference, and the P value is calculated by unpaired t-test;

[0024] Figure 7 The experimental results of (A) egg laying number and (B) hatching rate of Aedes albopictus in the Unloaded, dsNFS1 and CS-TPP-dsNFS1 groups. ns means no significant difference, and the P value is calculated by unpaired t test. DETAILED DESCRIPTION

[0025] Example 1

[0026] 1. Species tree construction and conserved sequence analysis

[0027] The 43 NFS1 amino acid sequences belonging to different kingdoms downloaded from NCBI were subjected to gene similarity analysis by blastp, and all NFS1 sequences with complete open reading frames were subjected to conservative motif analysis using the MEME (version 3.5.7) online server () with the following parameters: minimum width = 6, maximum width = 20, maximum number of motifs to be found = 7, and then the species tree was drawn and the conservative motifs were displayed using the () website. The results of AalbNFS1 similarity and conservative motif analysis are shown in Figure 1 .

[0028] 2. RNA extraction and gene cloning

[0029] Total RNA was extracted from the thorax of 30 female mosquitoes (Aedes albopictus) using TRIzol reagent (15596018CN, Invitrogen, USA) according to the instructions, and the concentration, purity and integrity of RNA were detected using Nanodrop 2000 spectrophotometer (NanoDrop Technologies, USA) and 1% agarose gel electrophoresis. One microgram of total RNA was reverse transcribed into cDNA in a 20 microliter reaction system using the TransScript One-Step gDNA Removal and cDNA Synthesis Kit (AT311-02, Transgen Biotech, China) for amplification of the target gene. PCR was performed using Golden Star T6 Super PCR Mix (TSE101, TsingKe Biotech, China) in a 50 μL reaction system containing 2.5 μL cDNA template, 2.5 μL upstream and downstream AalbNFS1 specific primers (forward primer: 5'-ACTCACCGCAGCGAAACT-3'; reverse primer: 5'-GGGTCGTCCACATCAAAGA-3') and 42.5 μL enzyme mixture. The amplification program for PCR amplification was as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 35 cycles; final extension at 72°C for 6 minutes. The PCR product was purified using the SanPrep column DNA gel extraction kit (B518131-0100, Sangon Biotech, China), which is the complete coding sequence of AalbNFS1. Its nucleotide sequence is shown in the sequence listing, SEQ ID NO: 1.

[0030] Example 2 Synthesis of dsNFS1

[0031] dsNFS1 (SEQ ID NO: 2) was synthesized in vitro using the T7 RiboMAX Express RNAi system (P1700, Promega, USA). Subsequently, dsNFS1 was purified using the MEGAclear™ kit (AM1908, Ambion, USA). The concentration of dsNFS1 was determined by the Nanodrop 2000 spectrophotometer as described above, and its integrity was analyzed on a 1% agarose gel.

[0032] Example 3 Synthesis of CS-TPP-dsNFS1 bionanosystem

[0033] First, a sodium tripolyphosphate (TPP) solution was prepared. A 1.0 mg / mL TPP (7758-29-4, Aladdin, China) solution was prepared with deionized water and the pH of the solution was adjusted to 4.5 by adding 0.1 M hydrochloric acid. Then, a 3.0 mg / mL chitosan (CS, 9012-76-4, Aladdin, China) solution was dissolved in 0.5% dilute acetic acid and stirred overnight at room temperature. The resulting solution was filtered using a 0.22 μm pore size filter to remove insoluble matter. The TPP solution was then coupled with CS and dsNFS1. Briefly, an appropriate amount of chitosan solution was added dropwise to an appropriate amount of TPP solution containing 35 μg of dsNFS1 under magnetic stirring at 600 rpm to ensure that the ratio of CS, TPP, and dsNFS1 in the reaction system was 5:1:0.0064. After 60 minutes of reaction, the reaction system was centrifuged at 12000 g for 5 minutes at 4°C, and the resulting precipitate was CS-TPP-dsNFS1 nanoparticles. The resulting precipitate was washed three times with deionized water, and finally dispersed in deionized water and stored in a refrigerator at 4°C for later use.

[0034] Example 4 Fluorescence quantitative PCR detection of the inhibitory effect of CS-TPP-dsNFS1 on AalbNFS1 expression

[0035] After Aedes albopictus were fed with beef liver powder (Unloaded group), beef liver powder with unloaded CS-TPP nanoparticles (CS-TPP group), beef liver powder with naked dsNFS1 (dsNFS1 group), and beef liver powder with CS-TPP-dsNFS1 nanoparticles (CS-TPP-dsNFS1 group) throughout their larval stages, pectoral muscle tissues of 30 female mosquitoes were obtained from the first to sixth days after the emergence of Aedes albopictus, and total RNA was extracted using TRIzol reagent (15596018CN, Invitrogen, USA). One microgram of total RNA was reverse transcribed into cDNA in a 20-microliter reaction using the TransScript One-Step gDNA Removal and cDNA Synthesis Kit (AT311-02, TransgenBiotech, China), and then quantitative PCR was performed using specific primers for the AalbNFS1 and AalbACTB genes (AalbNFS1 gene-specific primers: forward primer: 5′-GGGAGAAGGTTTCCGAGTG-3′; reverse primer: 5′-TTCACGGTCATTATGGACACTAG-3′), a LightCycler480 II 216 Detection System (Roche, China), and TransStart® Top Green qPCR SuperMix (AQ131, TransGen Biotech, China) under the following thermal cycling program: 94°C for 30 seconds; 94°C for 5 seconds, 55°C for 15 seconds, and 72°C for 10 seconds, for 45 cycles. The mRNA expression level of AalbNFS1 relative to that of AalbACTB gene was calculated using AalbACTB gene as internal reference gene (AalbACTB gene specific primers: forward primer: 5'-TGCTATGTCGCTCTTGACTTCG-3'; reverse primer: 5'-CGTTCGTTTCCAATGGTGATGA-3'). The data included three biological replicates, each of which contained three technical replicates. −ΔΔCt Methods The relative expression level was calculated. The quantitative results of the relative expression level of AalbNFS1 mRNA are shown in Figure 2 .

[0036] Example 5 Evaluation of the inhibitory effect of CS-TPP-dsNFS1 on the flight ability of Aedes albopictus

[0037] Aedes mosquitoes that were fed with beef liver powder (Unloaded group), beef liver powder with unloaded CS-TPP nanoparticles (CS-TPP group), beef liver powder with naked dsNFS1 (dsNFS1 group), and beef liver powder with CS-TPP-dsNFS1 nanoparticles (CS-TPP-dsNFS1 group) throughout the larval stage and 3 days after eclosion were gently placed on a cold petri dish with the dorsal side facing up after being fully anesthetized on ice. Figure 3 As shown, the tip of the tether (copper wire) was gently dipped into wood glue and then placed on the head of the Aedes albopictus and the angle between the tether and the mosquito body was approximately 30°, taking care to avoid interfering with wing vibrations. The fixed Aedes albopictus was then placed in a constant temperature environment (28°C) for 1 hour to allow it to fully recover from anesthesia. Next, the fixed Aedes albopictus was placed in the same direction and gently blown, and then immediately photographed with a camera for 10 minutes, recording the flight time and number of flights of each Aedes albopictus within 10 minutes. At least 5 Aedes albopictus were tested in each group, for a total of 10 sets of data. The inhibitory results of CS-TPP-dsNFS1 on the flight ability of Aedes albopictus are shown in Figure 2. Figure 4 .

[0038] Example 6 Evaluation of the inhibitory effect of CS-TPP-dsNFS1 on the blood-sucking ability of Aedes albopictus

[0039] Aedes mosquitoes fed with beef liver powder (Unloaded group), beef liver powder with unloaded CS-TPP nanoparticles (CS-TPP group), beef liver powder with naked dsNFS1 (dsNFS1 group), and beef liver powder with CS-TPP-dsNFS1 nanoparticles (CS-TPP-dsNFS1 group) throughout the larval stage and 4 days after eclosion were placed in a 50 × 50 × 50 cm cubic mosquito cage for at least 10 min to acclimate to the environment (28°C, 80% relative humidity). Figure 5 As shown, the powder-free latex gloves worn by the volunteers were cut into an opening with a diameter of about 50 mm, and the exposed skin was used as a blood meal for mosquitoes to suck. The time taken by the mosquito to land on the blood meal for the first time was recorded as the blood sucking response time. When it was full of blood, its blood sucking was observed through a stereo microscope (LeicaM165FC, Leica, Germany), and its blood sucking volume was calculated by the formula V=4 / 3πabc, where a, b, and c represent the ventral long axis, ventral short axis, and half of the lateral short axis, respectively. The inhibition results of CS-TPP-dsNFS1 on the blood sucking ability of Aedes albopictus are shown in Figure 6 .

[0040] Example 6 Evaluation of the inhibitory effect of CS-TPP-dsNFS1 on the reproductive capacity of Aedes albopictus

[0041] The female Aedes albopictus mosquitoes that had been tested for blood-sucking ability were kept separately in small cages with wet paper towels at the bottom according to the group. After 3 days, the mosquitoes were dissected in 1× phosphate buffer at room temperature, and the size and number of follicles in their ovaries were observed and measured under a stereo microscope (LeicaM165FC, Leica, Germany). The remaining female mosquitoes were allowed to lay eggs naturally and the number of eggs laid by each female mosquito on paper was counted, and the subsequent hatching rate calculation was based on this data. Ensure that 10 mosquitoes are measured for each indicator. The results of the inhibition of the reproductive ability of Aedes albopictus by CS-TPP-dsNFS1 are shown in Figure 2. Figure 7 .

[0042] Combine the following Figure 6 The results shown specifically illustrate the inhibitory effect of the blood-sucking ability of Aedes albopictus treated with the CS-TPP-dsNFS1 bio-nanosystem.

[0043] Aedes albopictus larvae were placed in a six-well plate containing 1 ml of deionized water. 50 μL of CS-TPP-dsNFS1 nanosystem containing 35 μg of dsNFS1 was mixed with 5 mg of beef liver powder (LA1040, Solarbio, China) and 100 μL of 1.5% melted agarose gel solution at 55°C. After the mixture was fully solidified, it was processed into 3 mm×3 mm×1 mm small round pieces. Approximately equal amounts of small round pieces were placed in deionized water and fed to larvae once a day until eclosion. The groups fed with only beef liver powder (Unloaded group), beef liver powder with unloaded CS-TPP nanoparticles (CS-TPP group), and beef liver powder with naked dsNFS1 (dsNFS1 group) were used as control groups. Four days after the mosquito larvae eclosion, the blood sucking response time, blood sucking frequency, and blood sucking amount of the four groups of mosquitoes were measured according to the method described above, and the data are expressed as mean ± standard deviation. For the obtained data, the Shapiro-Wilk normality test and variance test were first used to analyze whether the data followed a normal distribution and had homogeneous variance. When the data followed a normal distribution and had homogeneous variance, the unpaired t-test was used to analyze whether the difference between the two groups was significant using GraphPad Prism 8.0 (GraphPad Software, USA). P values ​​less than 0.05, 0.01, and 0.001 were considered to indicate significant (*), very significant (**), and extremely significant (***) differences between the two groups, respectively.

[0044] like Figure 6As shown in (A), (B) and (C), compared with the untreated beef liver powder (Unloaded group), beef liver powder with unloaded CS-TPP nanoparticles (CS-TPP group) and beef liver powder with naked dsNFS1 (dsNFS1 group), the blood-sucking response time of Aedes albopictus in the beef liver powder feeding group with CS-TPP-dsNFS1 (CS-TPP-dsNFS1 group) was significantly prolonged, the blood-sucking frequency was significantly reduced, and the blood-sucking volume was significantly reduced. These results all indicate that the CS-TPP-dsNFS1 bio-nanosystem can significantly inhibit the blood-sucking ability of Aedes albopictus.

Claims

1. A drug for controlling Aedes albopictus, characterized in that: The dsRNA targets the iron-sulfur cluster synthesis core protein AalbNFS1, and the nucleic acid sequence of the AalbNFS1 is shown in the sequence table SEQ ID NO:

1.

2. The drug for controlling Aedes albopictus according to claim 1, wherein: The nucleotide sequence of the dsRNA is shown in SEQ ID NO:

2.

3. A drug for controlling Aedes albopictus according to claim 1 or 2, characterized in that: dsNFS1, constructed into chitosan-sodium tripolyphosphate-dsNFS1 bionanomedicine.

4. The drug for controlling Aedes albopictus according to claim 3, wherein: The chitosan-sodium tripolyphosphate-dsNFS1 is prepared by the following method: 1) Prepare sodium tripolyphosphate TPP solution, and adjust the pH value to 4.0-5.0; 2) TPP solution is coupled with CS and dsNFS1; 3) Centrifuge, take out the precipitate and disperse.

5. The drug for controlling Aedes albopictus according to claim 4, wherein: The coupling described in step 3) is to add dsNFS1 and chitosan solution in sequence to the TPP solution under magnetic stirring at 600 rpm for reaction.

6. The drug for controlling Aedes albopictus according to claim 5, wherein: The weight ratio of chitosan, sodium tripolyphosphate and dsNFS1 in the bionano drug is 5:1:0.0064.

7. The drug for controlling Aedes albopictus according to claim 6, wherein: The centrifugation was performed at 12000 g for 5 minutes at 4°C. The precipitate was washed three times with deionized water, dispersed in deionized water, and stored in a refrigerator at 4°C.

8. The drug for controlling Aedes albopictus according to claim 7, wherein: The reaction time is 60 minutes.

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