Application of far-1 protein of brazzilian roundworm as an anti-nematode drug target

Abstract

The application discloses application of a Brazil roundworm FAR-1 protein as a nematode-resistant drug target point. The Brazil roundworm FAR-1 protein has the ability to bind to various fatty acids and retinol through a fluorescence ligand competition binding experiment; the Nbfar-1 gene is highly expressed in the whole development stage of the Brazil roundworm; the expression of the Nbfar-1 gene is inhibited through RNAi, which reduces the lipid content of the larvae, affects the egg laying rate of the adult, the egg hatching rate, the development of the larvae and the formation of the adult epidermis, and thus significantly affects the growth and development of the Brazil roundworm. Therefore, the Brazil roundworm FAR-1 protein can be used as a drug target point for screening or identifying a drug against parasitic nematodes, provides a method for development of the drug against parasitic nematodes, and has important significance for preventing and treating infection of the parasitic nematodes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of the FAR-1 protein of *Strombus haematobium* as a target for anti-nematode drugs. Background Technology

[0002] Hookworms, roundworms, and whipworms, common soil-transmitted intestinal parasitic nematodes, are widespread globally, posing a serious threat to human and various animal health. Currently available anti-nematode drugs have shown some resistance and exhibit varying degrees of side effects. There is an urgent need to identify key functional genes to develop more effective and low-toxicity anti-nematode drugs.

[0003] *Nippostrongylus brasiliensis* (Nb) is a soil-derived intestinal parasitic nematode that can induce an immune response similar to that of intestinal parasitic nematodes, especially hookworm infection. *Nippostrongylus brasiliensis* has become a common experimental animal model for screening anti-parasitic nematode drugs and studying infection immunity.

[0004] Fatty acid and retinol binding proteins (FARs) are a class of nematode-specific lipid-binding proteins that can bind fatty acids, retinol, retinoic acid, and phospholipids with high affinity, participating in the complex life processes of nematodes through the uptake and transport of lipids and retinol. However, the far gene in *Strombus brasiliensis* has not been studied, and its mechanism of regulating nematode development remains unclear. To provide more potential targets for anti-parasitic nematode drugs, it is necessary to study the function of the far gene in *Strombus brasiliensis*, an animal model for anti-nematode drug screening, during nematode development, which can provide a basis for screening drug targets for anti-parasitic nematode diseases. Summary of the Invention

[0005] This invention aims to provide the application of the FAR-1 protein of the Brazilian roundworm as a target for anti-nematode drugs.

[0006] The purpose of this invention is to provide the application of formulations that inhibit or silence the expression of fatty acid and retinol-binding protein FAR-1 or its encoding gene FAR-1 in *Strombus haematobium*.

[0007] Another object of the present invention is to provide an anti-nematode drug or a product that inhibits the growth of nematodes.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention demonstrates that the fatty acid and retinol-binding protein FAR-1 (NbFAR-1) of *Strombus haematobium* possesses the ability to bind various fatty acids and retinol, and is highly expressed throughout the entire developmental stage of the nematode. Interference with NbFAR-1 gene expression reduces the lipid content of larvae, affecting adult oviposition rate, egg hatching rate, larval development, and adult epidermal formation, thus significantly impacting the growth and development of *Strombus haematobium*. Furthermore, NbFAR-1 can serve as a drug target for screening and identifying anti-parasitic nematode drugs, providing a potential target for the development of anti-parasitic nematode drugs, which is of significant research value.

[0010] Furthermore, the amino acid sequence of the NbFAR-1 protein is shown in SEQ ID NO:1, and the nucleotide sequence of the Nbfar-1 gene encoding the protein is shown in SEQ ID NO:2.

[0011] Therefore, this invention provides the application of the fatty acid and retinol-binding protein FAR-1 of *Strombus haematobium* or its encoding far-1 gene as a drug target in screening or identifying drugs for nematode control.

[0012] This invention provides the use of formulations that inhibit or silence the expression of fatty acid and retinol-binding protein FAR-1 or its encoding gene FAR-1 in the nematode *Strombus brasiliensis* for the suppression of nematodes.

[0013] This invention provides the use of preparations of fatty acid and retinol-binding protein FAR-1 or its encoding Nbfar-1 gene from *Strombus brasiliensis* in the preparation of drugs for the prevention or treatment of nematode infections.

[0014] This invention provides the use of formulations that inhibit or silence the expression of fatty acid and retinol-binding protein FAR-1 or its encoding far-1 gene in the preparation of products that inhibit nematode growth.

[0015] This invention provides the application of a formulation that inhibits or silences the expression of fatty acid and retinol-binding protein FAR-1 or its encoding far-1 gene in the inhibition of nematode egg hatching.

[0016] This invention provides the use of formulations that inhibit or silence the expression of fatty acid and retinol-binding protein FAR-1 or its encoding far-1 gene in the preparation of products that inhibit the hatching of nematode eggs.

[0017] This invention provides an anti-nematode disease drug or a product that inhibits nematode growth, containing a formulation that inhibits or silences the expression of nematode fatty acid and retinol-binding protein FAR-1 or its encoding far-1 gene.

[0018] Preferably, the preparation is shRNA encoding the far-1 gene of fatty acid and retinol-binding protein from *Strombus haematobium*, or a recombinant vector or recombinant bacteria containing shRNA fragments.

[0019] More preferably, the nucleotide sequence of the shRNA precursor DNA is as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0020] Preferably, the nematode is *C. brevicornu*.

[0021] The present invention has the following beneficial effects:

[0022] This invention discloses the application of the fatty acid and retinol-binding protein FAR-1 or its encoding gene from *Strombus haematobium* (Brazilian nematode). This protein has the ability to simultaneously bind multiple fatty acids and retinol, and is highly expressed throughout the entire developmental stage of the nematode. Inhibiting Nbfar-1 gene expression reduces the lipid content of larvae, affecting adult oviposition rate, egg hatching rate, larval development, and adult epidermal formation, thus significantly impacting the growth and development of *Strombus haematobium*. Therefore, NbFAR-1 can serve as a drug target for screening or identifying drugs against parasitic nematodes, providing a target for the development of anti-parasitic nematode drugs and playing a significant role in the prevention and control of parasitic nematode infections.

[0023] This invention, through research on NbFAR-1 and worm development, aims to understand the mechanism by which the FAR-1 protein of *Strombus haematobium* affects the development of parasitic nematodes, providing potential targets for anti-parasitic nematode drugs and contributing to the development of drugs against parasitic nematode diseases. Attached Figure Description

[0024] Figure 1 Figure showing the ligand binding ability analysis results of NbFAR-1 protein (Note: *: P<0.05).

[0025] Figure 2 This is a graph showing the relative fluorescence spectra of retinol, NbFAR-1, and oleic acid. (Note: The horizontal axis represents wavelength, and the vertical axis represents relative fluorescence intensity.)

[0026] Figure 3 Expression profiles of the Nbfar-1 gene in *Sinocyclocheilus brasiliensis* at different developmental stages (Note: compared with the internal reference gene gapdh. *: P < 0.05, **: P < 0.01).

[0027] Figure 4 Nucleotide sequence information for the precursor DNA of shRNA-far-1-651 and shRNA-far-1-310 (Note: a is shRNA-far-1-651; b is shRNA-far-1-310).

[0028] Figure 5 Figure showing the changes in egg hatching, insect development, and L3 larval molting after Nbfar-1 gene RNAi (Note: **: P<0.01, ***: P<0.001).

[0029] Figure 6 Figure showing the morphological changes of insect eggs after Nbfar-1 gene RNAi (Note: **: P<0.01, ***: P<0.001).

[0030] Figure 7 Figure showing the morphological changes of L1 larvae after Nbfar-1 gene RNAi (Note: *: P<0.05, ***: P<0.001).

[0031] Figure 8 Figure showing the morphological changes of L2 larvae after Nbfar-1 gene RNAi (Note: *: P<0.05, ***: P<0.001).

[0032] Figure 9 Figure showing the morphological changes of L3 larvae after Nbfar-1 gene RNAi (Note: *: P<0.05, **: P<0.01, ***: P<0.001).

[0033] Figure 10 The figure shows the distribution and content of lipid droplets in L3 larvae after Nbfar-1 gene RNAi.

[0034] Figure 11 This image shows the morphological changes observed in adult insects after Nbfar-1 gene RNAi using scanning electron microscopy.

[0035] Figure 12 This image shows the morphological changes observed in L3 larvae after Nbfar-1 gene RNAi using scanning electron microscopy.

[0036] Figure 13 This image shows the changes in the ultrastructure of the adult insect epidermis after Nbfar-1 gene RNAi. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] The Brazilian roundworms used in the following examples were derived from nematodes preserved in the laboratory of this invention; the SPF-grade female SD rats used in the following experiments were all purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0040] Example 1: Analysis of the binding capacity of NbFAR-1 protein to fatty acids and retinol

[0041] This invention obtained a FAR protein FAR-1 (NbFAR-1) with high expression levels at various developmental stages of the Brazilian roundworm through cloning and sequencing analysis. Its amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence of the Nbfar-1 gene encoding the protein is shown in SEQ ID NO:2. Further functional analysis of NbFAR-1 was performed.

[0042] 1. The binding capacity of NbFAR-1 protein to various fatty acids

[0043] The binding affinity of NbFAR-1 protein to various fatty acids—saturated fatty acids (C16:0, C18:0), monounsaturated fatty acids (C18:1), and polyunsaturated fatty acids (C18:2, C20:4, C20:5)—was determined by a fluorescent ligand competitive binding assay.

[0044] Different experimental groups were set up in 96-well black microplates: (1) Protein control group: 10 μg NbFAR-1 protein; (2) Fatty acid analog DAUDA control group: 10 μM DAUDA; (3) DAUDA + NbFAR-1 protein group: 10 μg NbFAR-1 protein, 10 μM DAUDA; (4) DAUDA + NbFAR-1 protein + fatty acid: 10 μg NbFAR-1 protein, 10 μM DAUDA, 50 μM fatty acid. After thorough shaking, the microplates were placed in a multi-functional microplate reader, the excitation wavelength was set to 350 nm, the fluorescence values ​​at 380-700 nm were read, and the results were statistically analyzed.

[0045] The results showed that when NbFAR-1 protein and DUADA were excited by 345 nm light, their relative fluorescence intensity was weak when they existed alone, but significantly increased when they coexisted, peaking at 500 nm, indicating that NbFAR-1 protein has a strong binding affinity to DAUDA. In the NbFAR-1 protein and DAUDA coexisting system, the addition of 50 μM fatty acid significantly reduced the relative fluorescence intensity at the 500 nm peak, indicating that DAUDA bound to NbFAR-1 protein was displaced by fatty acid through competitive binding to NbFAR-1 protein. The relative fluorescence intensity was used to compare and analyze the preference of NbFAR-1 protein for different fatty acids, such as... Figure 1As shown, the NbFAR-1 protein exhibits good binding preference to C16:0, C18:0, C18:1, C18:2, C20:4, and C20:5, with the strongest binding preference to C18:1. In summary, these results indicate that the NbFAR-1 protein can bind to a variety of fatty acids.

[0046] 2. The binding capacity of NbFAR-1 protein to retinol

[0047] The binding ability of NbFAR-1 protein to retinol was detected by fluorescent ligand binding assay. Different experimental groups were set up in 96-well black ELISA plates: (1) protein control group: 10 μg NbFAR-1 protein; (2) retinol control group: 10 μM retinol; (3) retinol + NbFAR-1 protein group: 10 μg NbFAR-1 protein, 10 μM retinol; (4) retinol + NbFAR-1 protein + oleic acid group: 10 μg NbFAR-1 protein, 10 μM retinol, and different concentrations of oleic acid (1 μM, 2.5 μM, 5 μM, 10 μM). After thorough shaking, the plates were placed in a multi-functional ELISA reader, the excitation wavelength was set to 350 nm, and the fluorescence values ​​at 400-630 nm were read. The results were statistically analyzed.

[0048] The results showed that, under 350 nm excitation light, the relative fluorescence intensity of NbFAR-1 protein and retinol alone was relatively weak, such as... Figure 2 As shown, when both proteins coexist, a peak appears at 500 nm, indicating that the NbFAR-1 protein has the ability to bind to retinol. In the NbFAR-1 protein and retinol coexisting system, the addition of oleic acid resulted in a rightward shift of the peak and an increase in its peak value. Furthermore, increasing the concentration of oleic acid in the coexisting system showed that the peak value increased with increasing oleic acid concentration, indicating that the NbFAR-1 protein can simultaneously bind to oleic acid and retinol.

[0049] Example 2: Expression profile analysis of the Nbfar-1 gene at different developmental stages

[0050] 1. A total of seven body samples of *Strombus haematobium* from egg to adult stages were collected:

[0051] (1) Egg collection: Feces of infected SD rats were collected, and the feces containing eggs were purified by saline flotation method to obtain eggs;

[0052] (2) Collection of L1, L2 and L3 stage larvae: SD rat feces containing eggs were placed in a sieve and cultured at 26℃ in the dark for 24h, 48h and 7d to collect free L1, L2 and L3 stage larvae;

[0053] (3) Collection of L4 larvae: Infected L3 larvae were collected and injected subcutaneously into the abdomen of SD rats. On the third day after infection, the SD rats were euthanized, lung tissue was removed, and free L4 larvae were aspirated.

[0054] (4) Collection of L5 larvae and adults: Infected L3 larvae were collected and injected subcutaneously into the abdomen to infect SD rats. The SD rats were euthanized on days 6-7 and 12 after infection, and the intestines were removed. L5 larvae and adults were picked out under a stereomicroscope.

[0055] Total RNA was extracted from eggs, L1 larvae, L2 larvae, L3 larvae, L4 larvae, L5 larvae, and adults using the Simply P Total RNA Extraction Kit (Bio Flux). The RNA was then transferred using a reverse transcription kit (Prime Script). TM RT MasterMix reverse transcribes total RNA into cDNA.

[0056] 2. Real-time quantitative PCR detection

[0057] (1) Using cDNA from *Strombus brasiliensis* at different developmental stages as templates;

[0058] (2) RT-qPCR primer design: Primers were designed using Primer Premier 5.0 for the full-length sequence of the far-1 gene of *Strombus haematobium* (its nucleotide sequence is shown in SEQ ID NO:2) and the gapdh gene of *Strombus haematobium* (internal reference gene). Oligo 7.0 was used to analyze the relevant information of the sequences. The specific primer sequence information is as follows: far-1: F: 5'-GTTCTTAGCCAACAGTGTCTC-3'; R: 5'-GGTAACAAGCCAAACCTCG-3'; gapdh: F: 5'-GCAGCAGACGGACCAATG AAGG-3'; R: 5'-CACGAAGTTAGGGTTGAGCGAGATG-3';

[0059] (3) Using Light RT-qPCR was performed using a Roche 480 Instrument II instrument. The reaction system and procedure are shown in Tables 1 and 2 below.

[0060] Table 1 RT-qPCR Reaction System

[0061]

[0062] Table 2 RT-qPCR reaction procedure

[0063]

[0064] (4) After RT-qPCR detection is completed, use 2 -ΔΔCt Methods for analyzing relative expression levels;

[0065] The results are as follows Figure 3 As shown, the Nbfar-1 gene is highly expressed at all developmental stages, with relatively high expression in L4 and L5 larvae, exceeding the average expression level in other developmental stages by more than four times. The transcriptional level of this gene at different developmental stages indicates that the Nbfar-1 gene is relatively highly expressed during the parasitic stage.

[0066] Example 3: Effects of silencing the far-1 gene on the development of *Strombus haematobium* in Brazil

[0067] 1. siRNA synthesis

[0068] Based on the CDS and 3'UTR regions of the far-1 gene of *Strombus haematobium* in Brazil, siRNA interference fragments were designed. The siRNA sequence information is shown in Table 3. Two interference fragments were synthesized.

[0069] Table 3 siRNA sequence information

[0070]

[0071] 2. Evaluate the interference effect of siRNA on Nbfar-1 gene expression in in vitro cultured worms.

[0072] On day 11 after infection of SD rats, adult worms were collected from the rat intestines and counted. 500 μL of 10% DMEM complete medium, 50 worms, and 33 μg siRNA were added to each well of a 24-well cell culture plate. The plates were incubated at 37°C and 5% CO2 for 1 day, then flash-frozen in liquid nitrogen and stored at -80°C. Total RNA was subsequently extracted, reverse transcribed into cDNA, and detected by real-time quantitative PCR, using the gapdh gene as an internal control. The detection method was the same as in Example 3, and the results were statistically analyzed.

[0073] The results showed that the interference effects of the far-1-651 and far-1-310 fragments on Nbfar-1 gene expression reached approximately 30% and 42%, respectively.

[0074] 3. Recombinant lentivirus packaging preparation, titer determination and identification

[0075] (1) shRNA design: shRNA was designed based on the far-1-651 and far-1-310 interfering fragments. Single-stranded shRNA precursor DNA was synthesized and annealed to double strands. The interfering fragments shRNA-far-1-310 (sequence shown in SEQ ID NO: 3) and shRNA-far-1-651 (sequence shown in SEQ ID NO: 4), and the precursor DNA nucleotide sequence information are as follows: Figure 4 As shown;

[0076] (2) Plasmid construction: Restriction enzyme sites of BamHI and EcoRI were added to both ends of the shRNA precursor DNA, followed by double digestion of the pGIPZ vector plasmid and the shRNA precursor DNA. Ligation was performed using T4 ligase;

[0077] (3) Recombinant plasmid verification: The above PCR products were transformed into E. coli DH5α competent cells, and single clones were picked. The constructed lentiviral vector plasmids pGIPZ-far-1-651 and pGIPZ-far-1-310 were verified by bacterial PCR and first-generation sequencing. The results showed that the cloned sequences had no base differences. The verification primer sequences were: F: 5'-CAAGCCCGGTGCCTGAGTT-3'; R: 5'-TGGCCGGCCGCATTAGTCTT-3';

[0078] (4) Transfection: Using the liposome transfection reagent Lipofectamine 2000, the vector plasmid pGIPZ-far-1-651 / pGIPZ-far-1-310 and two helper plasmids psPAX2 and pMD2.G were transfected into HEK-293T cells and cultured overnight at 37°C and 5% CO2.

[0079] (5) Collecting the virus stock solution: 48-72 hours later, examine the expression of Turbo GFP (green fluorescent protein) in the cells using an inverted fluorescence microscope and take pictures; 48 hours after changing the medium, start collecting the cell supernatant;

[0080] (6) Concentration of virus stock solution: The virus stock solution was concentrated using the PEG8000-NaCl method;

[0081] (7) Determination of viral titer: The titer of recombinant lentivirus was determined using the Reed-Muench method.

[0082] 4. Evaluation of the interference effect of recombinant lentivirus on the Nbfar-1 gene of *Strombus haematobium* in Brazil

[0083] (1) Ectopicization treatment of L3 stage larvae: L3 stage larvae were treated with 0.2% sodium hypochlorite;

[0084] (2) Activation of L3 stage larvae: L3 stage larvae were enriched and counted, and cultured at 37℃ and 5% CO2 for 48-72h.

[0085] (3) L3 stage larvae and lentivirus co-incubation: Add concentrated lentivirus to the larval culture medium and continue culturing for 48-72h;

[0086] (4) RNAi effect stage: Remove the culture medium containing recombinant lentivirus, add fresh culture medium, and incubate at 37℃ for 48-72h;

[0087] (5) RNAi effect determination: insect bodies were collected, total RNA was extracted, reverse transcribed into cDNA, and RT-qPCR experiments were performed to evaluate the interference effect based on changes in Nbfar-1 gene expression.

[0088] 5. Effects of Nbfar-1 gene interference expression in *Strombus haematobium* on lipid droplet formation in L3 stage larvae.

[0089] Oil Red O staining of *Strombus haematobium*: Intestinal adult worms were collected, and eggs were obtained. L3 stage larvae were cultured using fecal culture, following the same method as step 1 in Example 2. The L3 stage larvae were then fixed with 1% paraformaldehyde. After repeated freeze-thaw cycles, the worms were washed with PBS buffer. 60% isopropanol was added for dehydration. The worms were then washed again with PBS buffer, and isopropanol Oil Red O solution (1% Triton X-100) was added for staining. The worms were observed and photographed using a stereomicroscope (Lecai), and measurements were performed using ImageJ software.

[0090] Lipids in L3 larvae after Nbfar-1 gene interference expression were detected using Oil Red O, such as... Figure 5 As shown, red lipid droplets are visible on both sides of the L3 stage larvae in the control group near the epidermis. The lipid droplet content of the 6th generation L3 larvae after continuous infection in the Nbfar-1 interference group is significantly reduced, indicating that the NbFAR-1 protein is involved in the formation of lipid droplets in the larvae.

[0091] 6. Effects of Nbfar-1 gene interference expression in *Strombus haematobium* on egg hatching, larval development, and L3 larval molting.

[0092] (1) Sampling: Female adult worms were collected from the intestines of SD rats 11 days post-infection, washed, and incubated at 37°C for 30 min. The adult worms were removed, all liquid was collected, the liquid was brought to a fixed volume, and the eggs were counted.

[0093] (2) Egg culture: The culture medium containing eggs is evenly distributed into the cell culture wells and cultured at 26°C in the dark.

[0094] (3) Development rate determination: After the eggs were cultured for 24h, 48h and 96h respectively, L1, L2 and L3 larvae were collected from the cell culture wells and counted and morphologically observed under an inverted microscope; Hatching rate = number of L1 stage larvae / number of eggs * 100%, L1-L3 stage larvae development rate = number of L3 stage larvae / number of eggs * 100%;

[0095] The results of observing the hatching of insect eggs and their development into larvae under in vitro culture conditions are as follows: Figure 6 As shown, the results indicated that the hatching rate of eggs in the Nbfar-1 interference group decreased by 19.87% and 13.08% (…). Figure 6 a) The rate of L1 larvae developing into L3 larvae decreased by 76.39% and 66.21%, respectively. Figure 6 b); After 8 days of in vitro culture, the molting rate of L3 stage larvae in the Nbfar-1 interference group was significantly higher than that in other groups, showing a phenomenon of premature molting of L3 stage larvae. Figure 6 c); After infecting rats with infective L3 stage larvae, the number of L3 stage larvae that could develop into adults decreased by 13.65% and 12.77% (c). Figure 6 d).

[0096] 7. Effects of Nbfar-1 gene interference expression on the morphology of eggs, L1, L2, L3 larvae, and adults of *Strombus haematobium*.

[0097] The eggs of parasites in the feces of infected rats were enriched by saline flotation. The eggs in the supernatant were examined under a microscope and then cultured in vitro. L1, L2 and L3 larvae in the culture dish were counted at 24, 48 and 96 h after culture. The images were taken and recorded using an inverted microscope. The images were processed using ImageJ. The length and width of the eggs and larvae were measured and the aspect ratio was calculated.

[0098] The effects of the Nbfar-1 gene on eggs of *Strombus haematobium* are as follows: Figure 7 As shown, the Nbfar-1 interference group exhibited abnormally shaped eggs, with enlarged cavities between the eggshell and blastomeres (a), and significantly reduced egg length (b) and length-to-width ratio (c). Observation of the morphology of L1-L2 stage larvae revealed... Figure 8-9 As shown, the L1-L2 larvae in the Nbfar-1 interference group are smaller, with significantly reduced length and width; further observation of the L3 larvae morphology reveals... Figure 10 As shown, the length and width of L3 stage larvae in the Nbfar-1 interference group increased slightly, while the length-to-width ratio decreased significantly, suggesting that interfering with Nbfar-1 gene expression can affect the size and morphology of eggs, L1, L2, and L3 stage larvae.

[0099] In summary, interfering with Nbfar-1 gene expression can affect lipid droplet formation, egg hatching rate, and larval development rate in L3 stage larvae, and the resulting larvae are smaller in size. This indicates that interfering with Nbfar-1 gene expression can effectively affect lipid acquisition, egg hatching, and larval development at all stages. This suggests that the FAR-1 protein in *Strombus haematobium* affects larval development through lipid binding and transport.

[0100] 8. Scanning electron microscopy (SEM) observation of the effects of far-1 gene interference expression on the morphology of L3 stage larvae and adults of *Strombus haematobium*.

[0101] (1) Prefixation: L3 stage larvae and rat intestinal worms were collected, washed repeatedly with PBS buffer, and fixed with 2.5% glutaraldehyde at room temperature.

[0102] (2) Refixation: Discard the 2.5% glutaraldehyde fixative, rinse the sample with PBS buffer, and fix the sample with 1% osmium tetroxide solution;

[0103] (3) Dehydration: Discard the 1% osmium tetroxide fixative and rinse the sample with PBS buffer; dehydrate the sample once each with ethanol solutions of gradient concentrations (30%, 50%, 70%, 80%, 90%), and then treat it twice with 100% ethanol solution.

[0104] (4) Critical point drying: The insect body is taken into a metal dish and placed in a carbon dioxide vacuum zero point dryer to remove the esters absorbed by the sample during the dehydration process.

[0105] (5) Coating and observation: The sample was placed on a metal glue and coated with 20nm gold particles. The external structure of the insect was observed using a scanning electron microscope (model: EVO MA 15) with a voltage of 5kV.

[0106] The morphology of L3 stage larvae was observed using scanning electron microscopy, such as... Figure 11 As shown, the L3 stage larvae in the Nbfar-1 interference group exhibited reduced cuticle roundness and swelling around the mouthparts on their heads. The morphology of the adults is as follows... Figure 12 As shown, the epidermis is loosely attached to the entire body surface of the worm, and the epidermis at the mouthparts is damaged. In summary, interference with Nbfar-1 gene expression causes abnormal epidermal morphology in L3 stage larvae and adults of *Strombus haematobium*.

[0107] 9. Observation of the effects of Nbfar-1 gene interference expression on the ultrastructure of adult insect epidermis using transmission electron microscopy (TEM).

[0108] (1) Sample collection and prefixation: Adult insects were collected, washed repeatedly with PBS buffer, and then fixed at room temperature with 2.5% glutaraldehyde solution.

[0109] (2) Buffer washing: Discard the 2.5% glutaraldehyde fixative and wash the sample four times with PBS buffer;

[0110] (3) Post-fixation: Discard the PBS buffer and add 1% hydroxyl tetraoxide fixative that can submerge the sample;

[0111] (4) Uranium acetate block staining: Discard the fixative and add a 50% ethanol saturated solution of uranium acetate;

[0112] (5) Gradient dehydration: Rinse four times with double-distilled water; then add 30%, 50%, 70%, 85%, and 95% ethanol in sequence for dehydration; finally add 100% ethanol twice for dehydration.

[0113] (6) Osmosis: Remove 100% ethanol, add acetone twice for transition; add propanol and resin mixture in sequence (ratio of 3:1, 1:1, 1:3), and let stand overnight after the last solution change;

[0114] (7) Resin embedding: Add pure resin twice;

[0115] (8) Encapsulation and polymerization: Polymerization in a 37℃ drying oven;

[0116] (9) Ultrathin sectioning: Slice the ultrathin section using an ultrathin slicer, and use a carbon support nickel mesh to pick up the ultrathin section onto filter paper and let it dry;

[0117] (10) Staining and observation: Stain with chloroform and observe and photograph under a transmission electron microscope (model: ThermoFisher Scientific Model Talos F200S);

[0118] Transmission electron microscopy was used to observe the morphology of the insects, such as... Figure 13 As shown, the Nbfar-1 interference group showed cavities in the inner fluid layer of the worm's epidermis, loose protrusions of the cortex outwards, irregular indentations of the basal layer inwards, thickening of the basement membrane, and the inability of the epidermal structure to maintain its original morphology.

[0119] In summary, this invention analyzed the ability of the NbFAR-1 protein to bind various fatty acids and retinol. Interfering with Nbfar-1 gene expression significantly reduced the lipid content of L3-stage larvae, decreased egg hatching rate and larval development rate. Further analysis of worm morphology showed that the epidermal formation of L3-stage larvae and adults was significantly affected, resulting in mouthpart epidermal damage and loosened epidermis in adults, preventing normal attachment to the body surface. Therefore, preparations used to silence or interfere with Nbfar-1 gene expression can be used to prepare drugs against nematodes. Simultaneously, NbFAR-1 can serve as a drug target for screening drugs against parasitic nematodes, providing a theoretical basis for the development of anti-parasitic nematode drugs and offering potential targets for these drugs, which is of significant research value.

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of an agent that inhibits or silences the expression of fatty acid and retinol binding protein FAR-1 or its encoding far -1 gene with fatty acid and retinol binding protein FAR-1 as a drug target for the preparation of a medicament for the treatment of nematode infections, characterized in that, The preparation is a coding gene of fat acid and retinol binding protein of Brazil roundworm far shRNA of -1 gene, or a recombinant vector, a recombinant bacteria or a recombinant virus containing the shRNA fragment; the nucleotide sequence of the shRNA precursor DNA is shown as SEQ ID NO: 3 or SEQ ID NO:

4. ​ 2. Formulations that inhibit or silence the expression of the fatty acid and retinol binding protein FAR-1 of B. xylophilus or its encoding far -1 gene in the preparation of products that inhibit the growth of nematodes, characterized in that, The preparation is a coding gene of fat acid and retinol binding protein of Brazil roundworm far shRNA of -1 gene, or a recombinant vector, a recombinant bacteria or a recombinant virus containing the shRNA fragment; the nucleotide sequence of the shRNA precursor DNA is shown as SEQ ID NO: 3 or SEQ ID NO:

4.

3. Use of an agent that inhibits or silences the expression of the fatty acid and retinol binding protein FAR-1 of B. runcatium or its encoding -1 gene in the preparation of a product for inhibiting hatching of nematode eggs, characterized in that, far -1 gene in the preparation of a product for inhibiting hatching of nematode eggs, characterized in that, The preparation is a coding gene of fat acid and retinol binding protein of Brazil roundworm far shRNA of -1 gene, or a recombinant vector, a recombinant bacteria or a recombinant virus containing the shRNA fragment; the nucleotide sequence of the shRNA precursor DNA is shown as SEQ ID NO: 3 or SEQ ID NO:

4.

4. The use according to any one of claims 1 to 3, characterized in that, The amino acid sequence of the fatty acid and retinol binding protein FAR-1 of the Brazilian roundworm is shown in SEQ ID NO: 1, which is encoded by the nucleotide sequence shown in SEQ ID NO:

2. far -1 gene is shown in SEQ ID NO: 2.

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