Application of Brazilian circular nematode FAR-1 protein as anti-nematode drug target

Abstract

The invention discloses an application of a Brazilian strongylus FAR-1 protein as an anti-nematode drug target. A fluorescent ligand competitive binding experiment shows that the Japanese strongylus Brazil FAR-1 protein has the capability of binding various fatty acids and retinol; the Nbfar-1 gene is in high expression in the whole development stage of the Brazilian strongylus; nbfar-1 gene expression is inhibited through RNAi, the lipid content of larvae can be reduced, the adult egg laying rate, the egg hatching rate, larva development and adult epidermis formation are affected, and therefore growth and development of Brazilian strongylus are remarkably affected. Therefore, the Brazilian circular nematode FAR-1 protein can be used as a drug target for screening or identifying anti-parasitic nematode drugs, a method is provided for development of the anti-parasitic nematode drugs, and the Brazilian circular nematode FAR-1 protein has important significance on prevention and treatment of infection of parasitic nematodes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to the application of the FAR-1 protein of Nippostrongylus brasiliensis as an anti-nematode drug target. Background Art

[0002] Common soil-transmitted intestinal parasitic nematodes such as hookworms, roundworms, and whipworms are widely prevalent globally and cause serious harm to the health of humans and various animals. At present, among the limited several anti-nematode drugs, some have developed drug resistance and have varying degrees of side effects. There is an urgent need to identify important functional genes in order to develop more anti-parasitic nematode drugs with high efficiency and low toxicity.

[0003] Nippostrongylus brasiliensis (Nb) is a soil-transmitted 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 anti-parasitic nematode drug screening and infection immunity research.

[0004] Fatty acid and retinol binding protein (FAR) is a class of nematode-specific lipid-binding proteins that can bind fatty acids, retinol, retinoic acid, and phospholipids with high affinity and participate in the complex life activities of nematodes by taking up and transporting lipids and retinol. However, there has been no research on the far gene of Nippostrongylus brasiliensis, and its mechanism of regulating nematode development remains undetermined. In order to provide more potential targets for anti-parasitic nematode drugs, it is necessary to study the function of the far gene of Nippostrongylus 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] The present invention aims to provide the application of the FAR-1 protein of Nippostrongylus brasiliensis as an anti-nematode drug target.

[0006] The object of the present invention is to provide the application of a preparation for inhibiting or silencing the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or the expression of its encoding far-1 gene.

[0007] Another object of the present invention is to provide an anti-nematode disease drug or a product for inhibiting nematode growth.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] The research of the present invention shows that the fatty acid and retinol binding protein FAR-1 (NbFAR-1) of Nippostrongylus brasiliensis has the ability to bind multiple fatty acids and retinol, and is highly expressed throughout the development stage of the nematode. After interfering with the expression of the Nbfar-1 gene, the lipid content of the larvae is reduced, affecting the egg-laying rate of adults, the hatching rate of eggs, the development of larvae, and the cuticle formation of adults, thus significantly affecting the growth and development of Nippostrongylus brasiliensis. At the same time, NbFAR-1 can be used as a drug target for screening and identifying anti-parasitic nematode drugs, providing a potential target for the development of anti-parasitic nematode drugs, and having important research significance.

[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, the present invention provides the application of the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or its encoding far-1 gene as a drug target in screening or identifying anti-nematode drugs.

[0012] The present invention provides the application of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or its encoding far-1 gene in inhibiting nematodes.

[0013] The present invention provides the application of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or its encoding Nbfar-1 gene in the preparation of drugs for preventing or treating nematode infections.

[0014] The present invention provides the application of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or its encoding far-1 gene in the preparation of products for inhibiting nematode growth.

[0015] The present invention provides the application of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or its encoding far-1 gene in inhibiting nematode egg hatching.

[0016] The present invention provides the application of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or its encoding far-1 gene in the preparation of products for inhibiting nematode egg hatching.

[0017] The present invention provides an anti-nematode drug or a product for inhibiting nematode growth, containing a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of nematodes or its encoding far-1 gene.

[0018] Preferably, the preparation is shRNA encoding the fatty acid and retinol binding protein far-1 gene of Nippostrongylus brasiliensis, or a recombinant vector or recombinant bacterium containing the shRNA fragment.

[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 Nippostrongylus brasiliensis.

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

[0022] The present invention discloses the application of the fatty acid and retinol binding protein FAR-1 of Nippostrongylus brasiliensis or its encoding far-1 gene. This protein has the ability to bind multiple fatty acids and retinol simultaneously and is highly expressed throughout the development stage of the nematode. After inhibiting the expression of the Nbfar-1 gene, the lipid content of the larvae is reduced, affecting the egg-laying rate, egg hatching rate, larval development, and cuticle formation of the adults of Nippostrongylus brasiliensis, thus significantly affecting the growth and development of Nippostrongylus brasiliensis. Therefore, NbFAR-1 can be used as a drug target for screening or identifying drugs against parasitic nematode diseases, providing a target for the development of anti-parasitic nematode drugs, which is of great significance for preventing and treating parasitic nematode infections.

[0023] Through the study of NbFAR-1 and worm development, the present invention understands the mechanism of action of the FAR-1 protein of Nippostrongylus brasiliensis in affecting the development of parasitic nematodes, provides potential action targets for anti-parasitic nematode drugs, and contributes to the research and development of drugs against parasitic nematode diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a result diagram for analyzing the ligand binding ability of the NbFAR-1 protein (Note: *: P<0.05).

[0025] Figure 2 It is a result diagram of the relative fluorescence spectrum when retinol, NbFAR-1, and oleic acid bind. (Note: The abscissa represents the wavelength, and the ordinate represents the relative fluorescence intensity).

[0026] Figure 3 It is an expression profile of the Nbfar-1 gene of Nippostrongylus brasiliensis at different development stages (Note: Compared with the internal reference gene gapdh. *: P<0.05, **: P<0.01).

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

[0028] Figure 5 Results of changes in egg hatching, worm development, and L3 larval sheath shedding after RNAi of the Nbfar-1 gene (Note: **: P<0.01, ***: P<0.001).

[0029] Figure 6 Results of changes in egg morphology after RNAi of the Nbfar-1 gene (Note: **: P<0.01, ***: P<0.001).

[0030] Figure 7 Results of changes in L1 larval morphology after RNAi of the Nbfar-1 gene (Note: *: P<0.05, ***: P<0.001).

[0031] Figure 8 Results of changes in L2 larval morphology after RNAi of the Nbfar-1 gene (Note: *: P<0.05, ***: P<0.001).

[0032] Figure 9 Results of changes in L3 larval morphology after RNAi of the Nbfar-1 gene (Note: *: P<0.05, **: P<0.01, ***: P<0.001).

[0033] Figure 10 Results of lipid droplet distribution and content in L3 larvae after RNAi of the Nbfar-1 gene.

[0034] Figure 11 Results of morphological changes observed by scanning electron microscopy in adults after RNAi of the Nbfar-1 gene.

[0035] Figure 12 Results of morphological changes observed by scanning electron microscopy in L3 larvae after RNAi of the Nbfar-1 gene.

[0036] Figure 13 Results of changes in the ultrastructure of the adult cuticle after RNAi of the Nbfar-1 gene. Detailed implementation methods

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

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

[0039] The Nippostrongylus brasiliensis used in the following examples is derived from the nematodes preserved in the laboratory of the present invention; the SPF-grade female SD rats used in the following experiments are all purchased from the Guangdong Provincial Medical Experimental Animal Center.

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

[0041] Through cloning and sequencing analysis, a FAR protein FAR-1 (NbFAR-1) with high expression levels at different developmental stages of Nippostrongylus brasiliensis was obtained. The 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 carried out.

[0042] 1. Binding ability of NbFAR-1 protein to various fatty acids

[0043] The binding ability of NbFAR-1 protein to various fatty acids: saturated fatty acids (C16:0, C18:0), monounsaturated fatty acid (C18:1), polyunsaturated fatty acids (C18:2, C20:4, C20:5) was detected by fluorescence ligand competition binding experiment.

[0044] Set different experimental groups in a 96-well black microplate: (1) Protein control group: 10 μg NbFAR-1 protein; (2) Fatty acid analogue 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 microplate was placed in a multifunctional microplate reader, the excitation wavelength was set at 350 nm, and the fluorescence values at 380 - 700 nm were read, and the results were statistically analyzed.

[0045] The results showed that when excited by 345 nm excitation light, the relative fluorescence intensity of NbFAR-1 protein and DUADA alone was weak, while the relative fluorescence intensity was significantly enhanced when the two coexisted, and a peak appeared at 500 nm, indicating that NbFAR-1 protein had a strong binding ability to DAUDA. In the coexistence system of NbFAR-1 protein and DAUDA, when 50 μM fatty acid was added, it was found that the relative fluorescence intensity at the 500 nm peak decreased significantly, indicating that DAUDA bound to NbFAR-1 protein was displaced by fatty acids through competitive binding to NbFAR-1 protein. Through the relative fluorescence intensity, the preference of NbFAR-1 protein for different fatty acids was compared and analyzed, such as Figure 1As shown, the NbFAR-1 protein has a good binding preference for C16:0, C18:0, C18:1, C18:2, C20:4, and C20:5, and has the best binding preference for C18:1. The above results show that the NbFAR-1 protein can bind multiple fatty acids.

[0046] 2. Binding ability of NbFAR-1 protein to retinol

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

[0048] The results showed that in the presence of 350 nm excitation light, when the NbFAR-1 protein and retinol existed alone, the relative fluorescence intensity was weak, as Figure 2 shown. However, when both coexisted, a peak appeared at 500 nm, indicating that the NbFAR-1 protein has the ability to bind retinol. In the coexistence system of the NbFAR-1 protein and retinol, when oleic acid was added, it was found that the peak shifted to the right and the peak value increased. On this basis, when the concentration of oleic acid in the coexistence system was increased, it was found that the peak value increased with the increase in the concentration of oleic acid, indicating that the NbFAR-1 protein can bind both oleic acid and retinol simultaneously.

[0049] Example 2 Analysis of the expression profile of the Nbfar-1 gene at each developmental stage

[0050] 1. A total of seven stages of worm samples of Nippostrongylus brasiliensis from eggs to adults were collected:

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

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

[0053] (3) Collection of L4-stage larvae: Collect infectious L3-stage larvae and infect SD rats by subcutaneous injection in the abdomen. On the 3rd day after infection, euthanize the SD rats, take out the lung tissues, and aspirate the free L4-stage larvae;

[0054] (4) Collection of L5-stage larvae and adults: Collect infectious L3 larvae and infect SD rats by subcutaneous injection in the abdomen. On the 6th - 7th day and the 12th day after infection of the SD rats, euthanize the SD rats respectively, take out the intestines, and pick out L5-stage larvae and adults under a stereomicroscope;

[0055] Use Simply P Total RNA Extraction Kit (Bio Flux) to extract the total RNA of eggs, L1-stage larvae, L2-stage larvae, L3-stage larvae, L4-stage larvae, L5-stage larvae, and adults, and use a reverse transcription kit (Prime Script TM RT MasterMix) to reverse transcribe the total RNA into cDNA.

[0056] 2. Real-time fluorescence quantitative PCR detection

[0057] (1) Use the cDNA of Nippostrongylus brasiliensis at different developmental stages as templates;

[0058] (2) Design of RT-qPCR primers: Use Primer Premier 5.0 to design primers for the full-length sequence of the far-1 gene of Nippostrongylus brasiliensis (its nucleotide sequence is shown in SEQ ID NO:2) and the gapdh gene (internal reference gene) of Nippostrongylus brasiliensis, and use Oligo 7.0 to analyze the relevant information of its sequence. 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) Use the Light 480 Instrument II (Roche) instrument to perform RT-qPCR detection. The reaction system and program are shown in Table 1 and Table 2 below;

[0060] Table 1 RT-qPCR reaction system table

[0061]

[0062] Table 2 RT-qPCR reaction program table

[0063]

[0064] (4) After the RT-qPCR detection, use 2 -ΔΔCt methods to analyze the change in relative expression level;

[0065] The results are as Figure 3 shown, indicating that the Nbfar-1 gene is highly expressed at all developmental stages, with relatively high expression in the L4 and L5 stage larvae, which is more than 4 times the average expression level of other developmental stages. From the transcriptional level of this gene at different developmental stages, the Nbfar-1 gene is relatively highly expressed during the parasitic life stage.

[0066] Example 3 Effect of silencing the far-1 gene on the development of Nippostrongylus brasiliensis

[0067] 1. siRNA synthesis

[0068] According to the CDS region and 3'UTR region of the far-1 gene of Nippostrongylus brasiliensis, siRNA interference fragments were designed. The siRNA sequence information is shown in Table 3, and 2 interference fragments were synthesized.

[0069] Table 3 siRNA sequence information

[0070]

[0071] 2. In vitro culture of worms to evaluate the interference effect of siRNA on the expression of the Nbfar-1 gene

[0072] On the 11th day after infecting SD rats, adult worms were obtained from the rat intestine and counted. A 24-well cell culture plate was selected, and 500 μL of 10% DMEM complete medium, 50 worms, and 33 μg of siRNA were added to each well. The culture plate was placed in an incubator at 37 °C and 5% CO 2 After culturing for 1 day, it was quickly frozen in liquid nitrogen and placed at -80 °C. Subsequently, total RNA was extracted, reverse transcribed into cDNA, and real-time fluorescence quantitative PCR detection was performed. Using the gapdh gene as an internal reference, 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 interference fragments on the expression of the Nbfar-1 gene reached about 30% and 42% respectively.

[0074] 3. Preparation, titer determination and identification of recombinant lentivirus packaging

[0075] (1) shRNA Design: Based on the far-1-651 and far-1-310 interfering fragments, design shRNA. Synthesize single-stranded shRNA precursor DNA and anneal it into double-stranded. The interfering fragments are shRNA-far-1-310 (the sequence is shown in SEQ ID NO: 3) and shRNA-far-1-651 (the sequence is shown in SEQ ID NO: 4). The nucleotide sequence information of the precursor DNA is as Figure 4 shown;

[0076] (2) Plasmid Construction: Add restriction enzyme cleavage sites of restriction enzymes BamH I and EcoR I to both ends of the shRNA precursor DNA, and then perform double digestion on the pGIPZ vector plasmid and the shRNA precursor DNA. Use T4 ligase for ligation;

[0077] (3) Verification of Recombinant Plasmid: Transform the above PCR products into E. coli DH5α competent cells, pick monoclonal bacteria, and verify the constructed lentiviral vector plasmids pGIPZ-far-1-651 and pGIPZ-far-1-310 by colony PCR and perform first-generation sequencing verification. The results show that there are no base differences in the cloned sequences. The verification primer sequences are: F: 5'-CAAGCCCGGTGCCTGAGTT-3'; R: 5'-TGGCCGGCCGCATTAGTCTT-3';

[0078] (4) Transfection: Use the liposome transfection reagent Lipofectamine 2000 to transfect the vector plasmids pGIPZ-far-1-651 / pGIPZ-far-1-310 and the two auxiliary plasmids psPAX 2 , pMD2.G into HEK-293T cells and culture them overnight at 37°C and 5% CO 2 conditions;

[0079] (5) Collection of Viral Stock Solution: After 48 - 72 hours, check the expression of Turbo GFP (green fluorescent protein) in cells by inverted fluorescence microscope and take pictures; 48 hours after changing the medium, start collecting the cell supernatant;

[0080] (6) Concentration of Viral Stock Solution: Concentrate the viral stock solution by the PEG8000-NaCl method;

[0081] (7) Determination of Viral Titer: Use the Reed-Muench method to determine the titer of the recombinant lentivirus.

[0082] 4. Evaluation of the Interference Effect of Recombinant Lentivirus Mediated by the Nbfar-1 Gene of Nippostrongylus brasiliensis

[0083] (1)Treatment for L3 larvae to shed the sheath: Treat L3 larvae with 0.2% sodium hypochlorite;

[0084] (2)Activation of L3 larvae: Enrich and count L3 larvae, and culture them at 37 °C and 5% CO 2 for 48 - 72 h;

[0085] (3)Co - incubation of L3 larvae and lentivirus: Add concentrated lentivirus to the worm culture medium and continue to culture for 48 - 72 h;

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

[0087] (5)Measurement of RNAi effect: Collect the worms, extract total RNA, reverse - transcribe it into cDNA, and perform RT - qPCR experiments. Evaluate the interference expression effect based on the change in the expression of the Nbfar - 1 gene.

[0088] 5. Effect of interfering expression of the Nbfar - 1 gene of Nippostrongylus brasiliensis on lipid droplet formation in L3 larvae

[0089] Oil Red O staining of Nippostrongylus brasiliensis: Collect adult worms from the intestine, obtain eggs, and culture L3 larvae through fecal culture method, the method is the same as step 1 of Example 2; Subsequently, fix L3 larvae with 1% paraformaldehyde; After repeatedly freezing and thawing the worms, wash the worms with PBS buffer; Add 60% isopropanol for dehydration treatment; Then wash the worms with PBS buffer, add isopropanol Oil Red O solution (1% Triton X - 100), stain, and observe and photograph with a stereomicroscope (Lecai), and measure and calculate using Image J software.

[0090] Detection of lipids in L3 larvae after interfering expression of the Nbfar - 1 gene using Oil Red O. As Figure 5 shown, it shows that red lipid droplet distribution can be seen in the regions near the epidermis on both sides of the body of L3 larvae in the control group. The lipid droplet content in the 6th generation of 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 worm body.

[0091] 6. Effect of interfering expression of the Nbfar - 1 gene of Nippostrongylus brasiliensis on egg hatching, larval development, and sheath shedding of L3 larvae

[0092] (1)Sampling: Obtain female adult worms from the intestine of SD rats 11 days after infection, wash them and incubate at 37 °C for 30 min. Pick out the adult worms, collect all the liquid, make the volume of the liquid constant and count the eggs;

[0093] (2) Egg culture: The culture solution containing eggs was evenly divided into cell culture wells and cultured in the dark at 26°C;

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

[0095] Under in vitro culture conditions, the results of egg hatching and development into larvae were observed as Figure 6 shown. The results showed that the egg hatching rate in the Nbfar-1 interference group decreased by 19.87% and 13.08% ( Figure 6 a), and the ratio of L1-stage larvae developing into L3-stage larvae decreased by 76.39% and 66.21% ( Figure 6 b); On the 8th day of in vitro culture, the molting rate of L3-stage larvae in the Nbfar-1 interference group was significantly higher than that of other groups, showing the phenomenon of premature molting of L3-stage larvae ( Figure 6 c); After infecting rats with infectious L3-stage larvae, it was found that the number of worm bodies that could develop into adults decreased by 13.65% and 12.77% ( Figure 6 d).

[0096] 7. Effects of interfering expression of Nbfar-1 gene of Nippostrongylus brasiliensis on the morphology of eggs, L1, L2, L3 stage larvae and adults

[0097] Eggs in the feces of infected rats were enriched by the saline flotation method, and the eggs in the supernatant were examined by microscopy and then cultured in vitro. After 24, 48, and 96 h of culture, the L1, L2, and L3 stage larvae in the culture dishes were counted, photographed and recorded under an inverted microscope, and the images were processed using Image J to measure the length and width of the eggs and larvae, and calculate the length-width ratio.

[0098] The results of the effect of Nbfar-1 gene of Nippostrongylus brasiliensis on eggs were as Figure 7 shown. Abnormally shaped eggs appeared in the Nbfar-1 interference group, the cavity between the eggshell and blastomeres became larger (a), and the egg length (b) and length-width ratio (c) were significantly reduced; Observing the morphology of L1-L2 stage larvae, as Figure 8 - 9 shown, it was shown that the morphology of L1-L2 larvae in the Nbfar-1 interference group became smaller, and the length and width were significantly reduced; Further observing the morphology of L3 stage larvae, as Figure 10 shown, the length and width of L3 stage larvae in the Nbfar-1 interference group slightly increased and the length-width ratio was significantly reduced, suggesting that interfering with the expression of Nbfar-1 gene can affect the size and morphology of eggs, L1, L2 and L3 stage larvae.

[0099] In summary, interfering with the expression of the Nbfar-1 gene can affect the lipid droplet formation, egg hatching rate, and larval development rate of L3-stage larvae. Moreover, the developed larvae are smaller in size, indicating that interfering with the expression of the Nbfar-1 gene can effectively affect lipid acquisition, egg hatching, and the development of larvae at all stages in the worm body. This suggests that the FAR-1 protein of Nippostrongylus brasiliensis affects worm development by binding and transporting lipids.

[0100] 8. Observation of the effects of interfering with the expression of the far-1 gene of Nippostrongylus brasiliensis on the morphology of L3-stage larvae and adults by scanning electron microscopy (SEM)

[0101] (1) Pre-fixation: Collect L3-stage larvae and adult worms in the rat intestine. After repeated washing with PBS buffer, fix them with 2.5% glutaraldehyde at room temperature.

[0102] (2) Re-fixation: Discard the 2.5% glutaraldehyde fixative, and rinse the samples with PBS buffer; fix the samples with 1% osmium tetroxide solution.

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

[0104] (4) Critical point drying: Aspirate the worms into a metal dish and place them in a carbon dioxide vacuum critical point dryer to remove the esters absorbed by the samples during dehydration.

[0105] (5) Coating and observation: Place the samples on metal glue, sputter coat them with 20 nm gold particles, and observe the external structure of the worms using a scanning electron microscope (model: EVO MA 15) with a voltage of 5 kV.

[0106] By observing the morphology of L3-stage larvae through SEM, as Figure 11 shown, the roundness of the epidermis of L3-stage larvae in the Nbfar-1 interference group decreased, and swelling was observed around the mouthparts of the head. The morphology of the adults is as Figure 12 shown, where it can be seen that the epidermis is loosely attached to the entire body surface of the worm, and the epidermis at the mouthpart is damaged. In summary, interfering with the expression of the Nbfar-1 gene causes abnormal epidermal morphology in L3-stage larvae and adults of Nippostrongylus brasiliensis.

[0107] 9. Observation of the effects of interfering with the expression of the Nbfar-1 gene on the ultrastructure of the adult worm epidermis by transmission electron microscopy (TEM)

[0108] (1) Sample collection and pre-fixation: Collect adult worms. After repeated washing with PBS buffer, fix them with 2.5% glutaraldehyde solution at room temperature.

[0109] (2) Buffer rinsing: Discard the 2.5% glutaraldehyde fixative and rinse the sample four times with PBS buffer.

[0110] (3) Post-fixation: Aspirate the PBS buffer and add 1% osmium tetroxide fixative that can submerge the sample.

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

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

[0113] (6) Infiltration: Aspirate the 100% ethanol and add acetone twice for transition; sequentially add a mixture of propanol and resin (in a ratio of 3:1, 1:1, 1:3), and let it stand overnight after the last liquid change.

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

[0115] (8) Embedding and polymerization: Polymerize in a 37°C air-drying oven.

[0116] (9) Ultra-thin sectioning: Section with an ultra-microtome, and pick up the ultra-thin sections with a nickel mesh supported by carbon glue and let them dry on filter paper.

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

[0118] The morphology of the worm body was observed by transmission electron microscopy. As Figure 13 shown, in the Nbfar-1 interference group, cavities appeared in the fluid layer inside the epidermis of the worm body, the cortex protruded loosely towards the outside of the body, the basal layer was irregularly sunken towards the inside of the body, the basement membrane thickened, and the epidermal structure could not maintain its original morphology.

[0119] In summary, the present invention analyzed that the NbFAR-1 protein has the ability to bind multiple fatty acids and retinol. After interfering with the expression of the Nbfar-1 gene, the lipid content of the L3-stage larvae was significantly reduced, and the egg hatching rate and larval development rate were decreased. Further analysis of the worm body morphology showed that the formation of the epidermis of the L3-stage larvae and adults was significantly affected, with damage to the epidermal mouthparts and loose cortex throughout the body of the adults, making it impossible to normally attach to the body surface. Therefore, the preparation for silencing or interfering with the expression of the Nbfar-1 gene can be used to prepare anti-nematode drugs. At the same time, NbFAR-1 can be used as a drug target for screening anti-parasitic nematode drugs, providing a theoretical basis for the development of anti-parasitic nematode drugs and potential action targets for anti-parasitic nematode drugs, which has important research significance.

[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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of the fatty acid and retinol binding protein FAR-1 of the Brazilian nematode or its encoding far-1 gene as a drug target in the screening or identification of drugs against parasitic nematodes.

2. Use of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 or its encoding far-1 gene in inhibiting nematodes.

3. Use of a preparation that inhibits or silences the expression of the fatty acid and retinol binding protein FAR-1 of the Brazilian nematode or its encoding far-1 gene in the preparation of drugs for preventing and treating nematode infections.

4. Use of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of the Brazilian nematode or its encoding far-1 gene in the preparation of a product for inhibiting nematode growth.

5. Use of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 or its encoding far-1 gene of the Brazilian nematode in inhibiting the hatching of nematode eggs.

6. Use of a preparation for inhibiting or silencing the expression of the fatty acid and retinol binding protein FAR-1 of the Brazilian nematode or its encoding far-1 gene in the preparation of a product for inhibiting the hatching of nematode eggs.

7. An anti-nematode drug or a product for inhibiting nematode growth, characterized in that: The invention discloses a preparation for inhibiting or silencing the expression of nematode fatty acid and retinol binding protein FAR-1 or its encoding far-1 gene.

8. The drug or product according to claim 7, characterized in that: The preparation is shRNA encoding far-1 gene of the fatty acid and retinol binding protein of the brasiliensis nematode, or a recombinant vector, recombinant bacteria or recombinant virus containing the shRNA fragment.

9. The drug or product according to claim 8, characterized in that: The nucleotide sequence of the shRNA precursor DNA is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

10. The use according to any one of claims 1 to 6, or the medicine or product according to claim 7, characterized in that: The amino acid sequence of the fatty acid and retinol binding protein FAR-1 of the brasiliensis nematode is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the far-1 gene is shown in SEQ ID NO: 2.

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