Method for intervening growth of echinococcus multilocularis based on diapause pheromone Ascr # 18 and application

Abstract

The invention discloses a method for intervening growth of echinococcus multilocularis based on diapause pheromone Ascr # 18 and application, and belongs to the technical field of prevention and control of zoonosis parasitic diseases. According to the invention, the Ascr # 18 is identified in the echinococcus multilocularis through liquid chromatography-mass spectrometry (LC-MS) for the first time, and it is verified that the Ascr # 18 can be combined with a downstream G-protein coupled receptor (GPCR) in a high-affinity manner, and a signal channel is activated. After the Ascr # 18 is treated, the expression level of development key genes daf-9 and daf-12 in the protoscolex of the echinococcus multilocularis can be remarkably reduced, and the movement activity of the development key genes daf-9 and daf-12 is inhibited, so that the development process and infection ability of parasites are blocked. The invention provides a novel biological prevention and control strategy which does not depend on traditional chemical anthelmintic drugs, is high in targeting, high in safety, environment-friendly and controllable, is suitable for comprehensive prevention and control of alveolar echinococcosis, and can also provide a theoretical basis and an application reference for prevention and control of other tapeworm and larva parasitic diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of parasitic prevention, and in particular to a method for interfering with the growth of multilocular hydatid based on diapause pheromone Ascr#18 and application thereof. BACKGROUND

[0002] Parasites are widely distributed in the world and pose a significant threat to human and animal health. Among them, trematodes, cestodes and nematodes belong to endoparasites, which often parasitize in the digestive tract, liver, lungs and other organs of the host, causing tissue damage, inflammation, secondary infection and other problems. These parasites not only seriously affect the growth and development and production performance of animals, but also can infect a variety of animals and humans, and their prevention and control have important veterinary and public health significance.

[0003] As an important group of Platyhelminthes, the adult of cestode mainly parasitizes in the intestinal tract of vertebrates, and the period of development in the intermediate host is called metacestode. The metacestode larvae mainly parasitize in the tissues and organs of the host, causing serious pathological damage. Echinococcus is one of the most clinically significant groups, and the hydatid disease caused by its larvae is listed as one of the 17 neglected tropical diseases in the world by the World Health Organization. According to different pathogen species, hydatid disease is mainly divided into cystic echinococcosis (caused by Echinococcus granulosus) and alveolar echinococcosis (caused by Echinococcus multilocularis).

[0004] Alveolar echinococcosis (AE) is a serious zoonotic parasitic disease caused by Echinococcus multilocularis larvae. The adult parasite lives in the intestinal tract of the final host, and the eggs are excreted with feces. Humans and rodents are infected by ingesting food or water contaminated with eggs, and the eggs develop into hydatid in the liver and other tissues of the intermediate host. When the final host preys on the tissues and organs containing hydatid, the protoscolex in the hydatid develops into adult in the intestinal tract of the final host, thereby completing its life cycle. Epidemiological data shows that AE is highly prevalent in Central Asia and western China.

[0005] The pathological features of multilocular echinococcosis are the formation of infiltrative space-occupying lesions in the liver, which can cause abdominal mass, pain, jaundice, and even liver failure. This disease has the characteristics of strong invasiveness, slow growth, and hidden symptoms, making early diagnosis difficult. When the disease progresses to the late stage, it can cause multiple organ metastasis, such as lung, brain, and bone, leading to systemic symptoms and threatening life. Currently, the main treatments for AE include surgical resection and conservative treatment with benzimidazole drugs (such as albendazole). However, surgical treatment has limitations such as high risk, high recurrence rate, etc., while drug treatment faces challenges such as poor efficacy, obvious side effects (such as liver function damage), and drug resistance. Long-term use of albendazole can cause serious systemic damage, and these treatment challenges need to be broken through. Therefore, developing new and efficient treatment options has become an important research direction in the field of AE prevention and treatment.

[0006] In recent years, the study of the molecular mechanisms of parasitic development regulation has provided important ideas for the development of new drugs. Among them, Ascarosides, a small molecule with unique signal regulation function, has attracted widespread attention. The function of Ascarosides was first studied in the model organism Caenorhabditis elegans. It can be recognized by G protein-coupled receptors (GPCRs) in the sensory neurons of nematodes, and then activate the conserved dauer signal pathway through second messengers such as cyclic guanosine monophosphate (cGMP), inducing the formation of dauer. Studies have shown that this signal pathway is highly conserved in many parasitic nematodes (such as Haemonchus contortus, Toxocara canis, and Strongyloides stercoralis). The key role of the DAF-9 (cytochrome P450, CYP450)–DA–DAF-12 (nuclear hormone receptor) module in the development transition and infection process of parasitic nematodes has been widely verified. In this conserved pathway, DAF-9 is a nematode-specific cytochrome P450 enzyme related to steroid hormone synthesis, mainly responsible for the synthesis of the steroid hormone ligand dafachronic acid (DA). DA can act as a ligand for DAF-12, regulate a series of gene expressions, and participate in key transitions in worm development decisions, such as dauer induction and exit, sexual maturation initiation, and tissue remodeling. In the absence of DA stimulation, DAF-12 binds to its co-repressor DIN-1, promoting the entry of worms into the dauer state; in the presence of DA, DAF-12 forms a transcriptional activation complex, promoting the continued development of worms. This regulatory model not only plays a key role in free-living nematodes, but also plays a similar 'development switch' role in many parasitic nematodes, participating in the transformation of larvae into infective stages, tissue invasion in the host, and the establishment of chronic infection. Therefore, the DAF-9–DA–DAF-12 module is one of the core pathways for parasitic worms to sense environmental signals, regulate their development, and adapt to hosts, and also provides a potential new intervention target for targeting the life cycle of parasitic worms.

[0007] At the same time, Ascarosides not only regulate the behavior and life cycle of parasites, but also have the potential to modulate the host immune system. Recent studies have found that Ascr#7 can significantly alleviate type II inflammatory response by inhibiting interleukin-33 (IL-33) production, reducing pathogenic type 2 helper T cells (Th2) and type 2 innate lymphocytes (ILC2), etc., showing application prospects for treating allergic diseases, and providing a non-living alternative strategy for live worm therapy. In the agricultural sector, Ascarosides can also induce plant innate immune responses, enhancing resistance to parasites and pathogens.

[0008] However, the existence and physiological function of diapause pheromones (Ascarosides) in tapeworms have not been reported, and this knowledge gap provides an important opportunity for original exploration of this research. Through tandem mass spectrometry MRM targeted detection technology, the present application first confirms the existence of Ascarosides #18 (Ascr #18) in the hydatid protoscolex, and further confirms that it may inhibit the development and infectivity of the protoscolex by regulating the conserved DAF-9-DA-DAF-12 homologous signal cascade reaction. This discovery not only fills the gap in the study of Ascarosides in tapeworm parasites, but also provides a new molecular basis for the development of new targeted drugs against echinococcosis.

[0009] Current methods for preventing and treating multilocular echinococcosis have significant limitations and cannot meet the needs of efficiency, safety, and sustainability. Chemical anthelmintic drugs (such as praziquantel and albendazole) are the main treatment method, but long-term use can induce parasite resistance, which does not meet the green concept of "reducing resistance and replacing resistance", the therapeutic effect decreases over time, and the side effects are toxic, therefore, chemical drugs have limited effect on the development and infectivity of the protoscolex, and it is difficult to effectively block the life cycle of the worm. Existing biological small molecules, such as Anacardic Acid, Nitazoxanide, small interfering RNA (siRNA), and exosome microRNA (miRNA), have certain anti-parasite potential, but face risks of toxicity (such as skin sensitization of Anacardic Acid), lack of in vivo validation (such as lack of animal experiment data for siRNA), technical complexity (such as poor stability of exosome delivery), and unclear efficacy, which limit clinical translation. In addition, there is currently no commercial vaccine for multilocular echinococcosis, and experimental vaccines (such as multiepitope vaccines) have not yet been implemented in clinical applications due to problems with antigen selection and immune persistence.

[0010] Currently, there is a lack of biological small molecules that can target the development and infectivity of echinococcosis. The purpose of the present application is to provide a small molecule intervention strategy based on worm-derived Ascarosides, which specifically inhibits the development and infectivity of the protoscolex by targeting the DAF-9-DA-DAF-12 signal cascade reaction of multilocular echinococcus, and blocks the life cycle of the worm. This strategy does not rely on chemical anthelmintic drugs, avoiding the risk of resistance and toxicity, and has the characteristics of high specificity, safety, and environmental friendliness, which is harmless to livestock, poultry, and humans, and meets the green ecological concept, providing a green and innovative biotechnology solution for the prevention and control of multilocular echinococcosis. SUMMARY

[0011] The present application aims at the deficiencies of the prior art, and provides a method for interfering with the growth of multilocular hydatid based on diapause pheromone Ascr#18 and application thereof, based on identification and use of a natural chemical small molecule Ascaroside existing in the multilocular hydatid, the following solution is proposed: firstly, mass spectrometry is used to prove for the first time that Ascr#18 exists in the multilocular hydatid, and a potential G protein-coupled receptor GPCR is identified by means of molecular docking technology, which proves that it has signal transduction potential; then, qPCR experiments prove that the expression of key genes daf-9 and daf-12 in the dauer signal pathway can be significantly down-regulated after the protoscolex is treated by Ascr#18, so as to interfere with the development regulation pathway; at the same time, the activity of the protoscolex is significantly reduced after being treated by Ascr#18 through protoscolex activity determination. Based on this, the present application uses Ascr#18 to act on the GPCR-DAF-9-DAF-12 signal axis of the parasite, targets to inhibit the development and infection process of the protoscolex of the multilocular hydatid, so as to block the life cycle, and provides a new type of biological prevention and control strategy with high specificity, safety and environmental protection. This scheme not only responds to the advocacy of “reducing and banning antibiotics”, but also provides a new technical path for the comprehensive prevention and control of multilocular (vesicular) hydatid disease.

[0012] The purpose of the present application is achieved by the following technical scheme: a method for interfering with the growth of multilocular hydatid based on diapause pheromone Ascr#18, adding exogenous Ascr#18, which can bind to the G protein-coupled receptor GPCR of the multilocular hydatid, down-regulate the expression level of the downstream daf-9 / daf-12 gene, and inhibit the development and infection activity of the protoscolex of the multilocular hydatid.

[0013] Further, the natural small molecule Ascr#18 existing in the protoscolex of the multilocular hydatid can bind to the GPCR receptor as an endogenous signal molecule of the parasite, transmit signals and participate in the regulation of the downstream development process.

[0014] Further, the GPCR-daf-9-daf-12 signal axis of the parasite is used to target the development and infection process of the protoscolex of the multilocular hydatid, so as to block the life cycle.

[0015] Further, with the increase of the concentration of the exogenous Ascr#18, the inhibition effect of the development and infection activity of the protoscolex of the multilocular hydatid is enhanced.

[0016] Further, the fluorescence quantitative PCR primers for the daf-9 and daf-12 genes are designed to analyze the gene expression changes of the multilocular hydatid after being treated by the exogenous Ascr#18.

[0017] Further, the qPCR primers of the daf-9 gene are as follows: an upstream primer: SEQ ID NO. 1: 5'-AATGTGGTGAGCCCGTAAA-3'; a downstream primer: SEQ ID NO. 2: 5'-AGCAGTTCGTCAACCGTATC-3'.

[0018] Further, the qPCR primers of the daf-12 gene are as follows: an upstream primer: SEQ ID NO. 3: 5'-TGTTCGCCTTGTCGTCTTT-3'; a downstream primer: SEQ ID NO. 4: 5'-CTGAATCTTCTCCCTTCGTTCTC-3'.

[0019] In a second aspect, the present application further provides an application of the dauer pheromone Ascr#18 in inhibiting the motility activity of the protoscoleces of multilocular echinococcosis.

[0020] In a third aspect, the present application further provides an application of the dauer pheromone Ascr#18 in preparing a biological preparation for preventing and treating alveolar echinococcosis (AE).

[0021] Advantages of the present application:

[0022] The present application is a biological technical solution which can effectively block the development and infection activity of multilocular echinococcosis without relying on chemical deworming drugs. The solution is based on the process of "signal sensing-development regulation-infection establishment" of multilocular echinococcosis, and for the first time uses the small molecule pheromone Ascr#18 naturally existing in the body as a core intervention factor to target regulate the GPCR-daf-9-daf-12 signal axis, so as to achieve the purpose of blocking the development and infection ability of the larvae, and has good targeting and biological safety.

[0023] Among them, Ascr#18 is confirmed by liquid chromatography-mass spectrometry (LC-MS) as a small molecule endogenous signal of parasites; through molecular docking prediction, it can be combined with G protein-coupled receptor GPCR with high affinity and mediate signal transmission; Ascr#18 treatment can significantly down-regulate the expression levels of the key genes daf-9 and daf-12 in the protoscoleces; the movement ability of the protoscoleces treated under in vitro culture conditions is significantly reduced, indicating that the infection ability is significantly weakened. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0025] Figure 1 is the Ascr#18 mass spectrum identification figure.

[0026] Figure 2 is the Ascr#18 and G protein-coupled receptor GPCR molecular docking and binding energy schematic diagram.

[0027] Figure 3 is the schematic diagram of the activity detection of the protocorm after being treated with different concentrations of Ascr#18 for 24h.

[0028] Figure 4 is the schematic diagram of the daf-9 and daf-12 gene transcription level of the protocorm after being treated with different concentrations of Ascr#18 for 24h. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and points of the present application more clear, the present application will be further described below in combination with the drawings and implementation examples. It should be understood that the specific implementation examples described herein are only used to explain the present application, and are not used to limit the present application.

[0030] The present application aims at the current situation of lacking biological small molecules targeting the activity of parasite development and infection in the prevention and control of Echinococcus multilocularis infection, and proposes an innovative intervention strategy based on natural small molecule Ascaroside (Ascr#18). Ascaroside small molecules, as nematode-derived diapause pheromones, are known to be able to regulate various developmental processes of nematodes, suggesting that Ascr#18 may have similar signal molecule functions in Echinococcus multilocularis.

[0031] Currently, Ascarosides are widely studied in free-living nematodes (such as Caenorhabditis elegans), and there is no systematic identification in Echinococcus multilocularis. The present application uses liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry) to separate and identify Ascr#18 from Echinococcus multilocularis in vitro culture extract. The experiment uses a high-resolution mass spectrometer (model: Q Exactive Orbitrap), and by comparing the mass spectrum with the known standard spectrum, it is confirmed that the molecular structure (molecular weight, fragment ion mode) of Ascr#18 is consistent with the literature report. The results show that Ascr#18 exists in the protocorm of Echinococcus multilocularis. It is an endogenous signal molecule in the parasite and can participate in the regulation of downstream development. The present application first confirms the existence of Ascr#18 in Echinococcus multilocularis, which lays a foundation for subsequent functional research. Figure 1 ​

[0032] Secondly, the binding affinity of Ascr#18 to the hydatid G protein-coupled receptor GPCR was predicted by using the molecular docking technique based on structural biology (software: AutoDock Vina 1.1.2). The three-dimensional prediction of the hydatid GPCR protein was performed by the AlphaFold 3 website to simulate the interaction of Ascr#18 with the receptor binding site Figure 2 : schematic diagram). The docking results showed that Ascr#18 had a high binding affinity to a specific GPCR receptor (sequence ID: EmuJ_001171500) (binding energy -7.7 kcal / mol), indicating that it might transmit signals through GPCR Figure 2 ).

[0033] To clarify the specific role and mechanism of Ascr#18 in hydatid, the hydatid protoscoleces were isolated from Meriones unguiculatus, evenly plated in 24-well plates and added with DMEM culture medium, and cultured in vitro at 37℃ in a 5% (v / v) CO2 incubator. After 24h treatment with Ascr#18 (concentration: 100nM, 10μM), the activity of the protoscoleces was determined by using the worm activity meter (wMicroTracker ONE) for 30min, and the activity distance and suction cup contraction frequency of the protoscoleces per minute were recorded. The results showed that the activity of the protoscoleces in the Ascr#18 treatment group was significantly reduced, and the inhibitory effect was enhanced with increasing concentration, indicating that the activity of the protoscoleces was reduced after the addition of Ascr#18, indicating that the infectivity was inhibited Figure 3 ).

[0034] Meanwhile, real-time fluorescent quantitative PCR technology was used to design qPCR primers for daf-9 (cytochrome P450, CYPs) and daf-12 (nuclear hormone receptor, Nuclear hormone receptor) genes (daf-9: upstream primer: SEQ ID NO. 1: 5'-AATGTGGTGAGCCCGTAAA-3'; downstream primer: SEQ ID NO. 2: 5'-AGCAGTTCGTCAACCGTATC-3'; daf-12: upstream primer: SEQ ID NO. 3: 5'-TGTTCGCCTTGTCGTCTTT-3'; downstream primer: SEQ ID NO. 4: 5'-CTGAATCTTCTCCCTTCGTTCTC-3'), and the gene expression changes of E. granulosus after Ascr#18 addition treatment were analyzed. The protoscoleces were collected and the total RNA of the worms was extracted. The gene expression levels of daf-9 and daf-12 were detected by qPCR (instrument: LightCycler® 480 Instrument II). The results showed that compared with the blank control, the expression levels of daf-9 and daf-12 in the Ascr#18 treatment group were significantly down-regulated (P < 0.05), which indicated that Ascr#18 inhibited the development signal pathway of the parasite at the transcriptional level Figure 4 ).

[0035] In summary, the Ascr#18 in E. granulosus is identified by mass spectrometry analysis in the present application, the binding with GPCR receptors is verified by means of protein structure modeling, molecular docking, etc., the inhibition of protoscoleces development and infection activity by down-regulating the expression levels of daf-9 / daf-12 is verified by activity determination and qPCR experiments, and a kind of efficient, specific and safe biological prevention and control strategy is constructed. The scheme overcomes the limitations of chemical drug resistance, toxicity and existing biological small molecule transformation difficulties, and provides an innovative biological technology path for the comprehensive prevention and control of E. granulosus disease.

[0036] The application identifies a natural small molecule Ascr#18 in hydatid cysts for the first time by liquid chromatography-high resolution mass spectrometry, performs molecular docking of Ascr#18 and hydatid cyst G protein-coupled receptor (GPCR), and indicates that Ascr#18 has the potential to transmit downstream signals. Based on this, qPCR primers for daf-9 and daf-12 are designed, and the gene expression of protoscoleces after Ascr#18 treatment is detected, and the results show that the expressions of the two key development genes are significantly down-regulated, which confirms that Ascr#18 can inhibit the development of hydatid cysts through the GPCR-daf-9-daf-12 signal axis. The activity of protoscoleces is determined to evaluate the influence of Ascr#18 on the movement ability of protoscoleces, and it is found that Ascr#18 can slow down the movement speed and contraction frequency of protoscoleces in a concentration-dependent manner, which indicates that Ascr#18 effectively reduces the infectivity of hydatid cysts.

[0037] The application identifies a natural pheromone Ascr#18 in hydatid cysts based on liquid chromatography-mass spectrometry (LC-MS), confirms that Ascr#18 can be combined with GPCR with high affinity through molecular docking, has signal transmission potential, and further confirms that Ascr#18 can down-regulate the gene expression of the daf-9 / daf-12 development pathway and significantly inhibit the movement ability of protoscoleces through qPCR and protoscolex activity detection, thereby confirming that Ascr#18 plays a key role in inhibiting the development and infectivity of hydatid cyst protoscoleces.

[0038] Example 1 Extraction and identification of Ascr#18 of hydatid cyst

[0039] (1) Isolation and purification of protoscoleces

[0040] The hydatid cysts adhered to the abdominal cavity and tissues of the passaged gerbils were taken out, the cyst tissue was cut with scissors and placed on a 180-micron filter screen, and 1x penicillin-streptomycin (PS) containing PBS buffer was continuously added during the process. A syringe rubber stopper was used for grinding. The protoscolex suspension filtered by the filter screen was collected, PBS solution was added for several times for standing, the supernatant was discarded and the precipitate was collected, and the filtration collection step was repeated for about 5 times. The protoscolex mixture precipitate was visible.

[0041] (2) Mass spectrometry sample pretreatment

[0042] 500 μL of methanol and 2 steel balls were added to the sample tube, and grinding was performed twice, each time for 3 min, and then centrifugation was performed for 15 min (4 ℃, 20000 rpm), the supernatant was transferred to a new EP tube, and after freezing and drying, 200 μL of methanol was used for redissolution, and then centrifugation was performed for 15 min (4 oC, 20000 rpm), and the supernatant was taken to an LC vial for LC-MS / MS analysis.

[0043] (3) Mass spectrometry instrument analysis

[0044] The chromatographic condition parameters include a chromatographic column (Agilent EC-C18; 2.1x100 mm, 2.7 μm), a column temperature (30 ℃), an injection volume (20 μL), a mobile phase (A: 5 mmol / L ammonium fluoride aqueous solution; B: acetonitrile), and mass spectrometry condition parameters are a multiple reaction monitoring mode (MRM), a negative ion mode (Negative), an ion source is an electrospray ionization source (ESI), an ion source temperature (TEM) 400 ℃, a spray voltage (IS) -4500 V in a negative ion mode, and a curtain gas (CUR) 35 psi.

[0045] The detection results are shown in Table 1. Figure 1

[0046] Example 2 Verification of Echinococcus multilocularis Ascr#18 as a quiescence signal molecule

[0047] Ascr#18 and GPCRs receptor molecule docking screening

[0048] The three-dimensional protein structure of Echinococcus multilocularis GPCRs was obtained in advance by AlphaFold3 simulation. The protein was pretreated by means of the "Prepwiz" module of the software Schrödinger. That is, the target protein is converted into a form that can be docked with the ligand by modeling calculation, bond order assignment, charge distribution, addition of hydrogen atoms, manufacture of missing side chains or ring atoms.

[0049] The chemical structure of Ascr#18 was downloaded from pubchem. By means of the "LigPrep" module of the software Schrödinger, the ligand file was converted into a three-dimensional structure with the lowest energy, and standardized processing such as hydrogenation, charging, and generating molecular conformation was carried out, so as to facilitate subsequent docking.

[0050] The prepared GPCRs protein was used as the receptor, and the Ascr#18 molecule was used as the ligand. The software AutoDock Vina1.1.2 was used for protein-molecule docking, and a docking score table was output. The software PyMOL V2.5 (Schrödinger company) was used for visual analysis of the docking results. The results are shown in Table 2. Figure 2

[0051] Example 3 Effect of exogenous Ascr#18 addition on the infectivity of protoscoleces

[0052] ​​Activity assay of protoscolex after Ascr#18 addition treatment

[0053] Uniformly plated into 24-well plates and added with DMEM culture solution, in vitro culture was carried out at 37°C in a 5% (volume fraction) CO2 incubator, after 24h treatment of Ascr#18 (concentration: 100nM, 10μM), the activity of protoscolex was determined by using the worm activity meter (wMicroTracker ONE), the determination time was 30min, and the activity distance and suction cup contraction frequency of protoscolex per minute were recorded.

[0054] The obtained activity values were input into Microsoft Excel for relative quantitative analysis, t test method and one-way analysis of variance method were used for significance analysis, and software GraphPad Prism 9.5 was used for plotting, and the results are shown in Figure 3 .

[0055] Example 4 Influence of exogenous Ascr#18 addition on the development of protoscolex

[0056] (1) In vitro culture of protoscolex

[0057] Uniformly plated into 24-well plates and added with 500μl of 1× penicillin-streptomycin DMEM culture solution, in vitro culture was carried out at 37°C in a 5% CO2 (volume fraction) incubator, after 24h treatment of Ascr#18 (concentration: 100nM, 10μM), the worm bodies of the blank group and the experimental treatment group were collected.

[0058] (2) Total RNA extraction and reverse transcription reaction

[0059] RNeasy Plus Mini Kit kit (Qiagen) was used for RNA extraction of each group of protoscolex. An appropriate amount of RNase-free water was added to dissolve the precipitate, and micro-nucleic acid analyzer and RNA gel electrophoresis were used for quality inspection, then 1 microgram of RNA was used for reverse transcription using FSQ-301 reagent.

[0060] (3) Fluorescent quantitative PCR

[0061] The Em-β-actin gene was used as an internal reference, and specific primers were used for real-time fluorescent quantitative PCR (qRT-PCR) reaction.

[0062] The qPCR reaction system was 10 μL ChamQ Universal SYBR qPCR Master Mix, 0.4 μL upstream primer, 0.4 μL downstream primer, 1 μL cDNA template, and 8.2 μL RNase-free water. The qPCR reaction condition was pre-denaturation at 95℃ for 30 sec, cycle reaction (95℃ for 10 sec, 60℃ for 10 sec, 40 cycles), and melting curve reaction condition was 95℃ for 15 sec, 60℃ for 60 sec, and 95℃ for 15 sec.

[0063] The obtained cycle threshold (CT value) was compared by Microsoft Excel with 2 -ΔΔCT The relative quantitative analysis was carried out by t test method and single factor variance analysis method, and the software GraphPad Prism 9.5 was used for drawing to analyze the transcription level difference of daf-9 and daf-12 genes under the condition of adding different concentrations of Ascr#18, and the results are shown in Figure 4

[0064] The above examples are used to explain and illustrate the present application, but not to limit the present application, any modification and change made to the present application within the spirit and protection scope of the claims of the present application, falls into the protection scope of the present application.​

Claims

1. A method for interfering with the growth of Echinococcus multilocularis based on the use of the Daphnia magna pheromone Ascr#18, characterized in that, Add exogenous Ascr#18, which can bind to GPCR, the multilocular hydatid G protein-coupled receptor, to down-regulate the expression level of downstream daf-9 / daf-12 genes and inhibit the development and infection activity of multilocular hydatid protoscoleces.

2. The method for interfering with the growth of Echinococcus multilocularis based on diapause pheromone Ascr#18 according to claim 1, characterized in that, The presence of Ascr#18 in multilocular hydatid protoscoleces As a parasitic endogenous signal molecule, Ascr#18, a natural small molecule, can bind to GPCR receptors to transmit signals and participate in the regulation of downstream development processes.

3. The method for interfering with the growth of Echinococcus multilocularis based on diapause pheromone Ascr#18 according to claim 1, characterized in that, By targeting the GPCR-daf-9-daf-12 signaling axis of Ascr#18 acting on parasites, the development and infection process of multilocular hydatid protoscoleces are inhibited, thereby blocking the life cycle.

4. The method for interfering with the growth of Echinococcus multilocularis based on diapause pheromone Ascr#18 according to claim 1, characterized in that, With the increase of the concentration of exogenous Ascr#18, the inhibitory effect of multilocular hydatid protoscoleces development and infection activity is enhanced.

5. The method for interfering with the growth of Echinococcus multilocularis based on diapause pheromone Ascr#18 according to claim 1, characterized in that, Design real-time fluorescent quantitative PCR primers for daf-9 and daf-12 genes to analyze the gene expression changes of multilocular hydatid after exogenous Ascr#18 addition treatment.

6. The method for interfering with the growth of Echinococcus multilocularis based on diapause pheromone Ascr#18 according to claim 5, characterized in that, The qPCR primer for daf-9 gene is: upstream primer: SEQ ID NO. 1: 5'-AATGTGGTGAGCCCGTAAA-3'; downstream primer: SEQ ID NO. 2: 5'-AGCAGTTCGTCAACCGTATC-3'.

7. The method for interfering with the growth of Echinococcus multilocularis based on diapause pheromone Ascr#18 according to claim 5, characterized in that, The qPCR primer for daf-12 gene is: upstream primer: SEQ ID NO. 3: 5'-TGTTCGCCTTGTCGTCTTT-3'; downstream primer: SEQ ID NO. 4: 5'-CTGAATCTTCTCCCTTCGTTCTC-3'.

8. Application of diapause pheromone Ascr#18 in inhibiting the motility activity of multilocular hydatid protoscoleces.

9. Application of diapause pheromone Ascr#18 in preparing biological agents for preventing and treating alveolar echinococcosis AE.

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