A siRNA for intervening the sex ratio of the Haemonchus contortus population and its application

Abstract

The present invention discloses a siRNA for intervening the sex ratio of a Haemonchus contortus population and its application, and relates to the field of animal parasitic disease prevention and treatment. The present invention has confirmed in vivo experiments in ruminant Hu sheep that the daily ovulation amount of the attacking group after applying the siRNA primer of the present invention is significantly reduced, the ovulation peak is delayed by 4 days compared with the normal group, and the insect load of adults (especially females) is significantly reduced, which plays a certain role in blocking transmission.

Description

Technical Field

[0001] The present invention relates to the field of animal parasitic disease prevention and treatment, and in particular to a siRNA for intervening in the sex ratio of a Haemonchus contortus population and its application. Background Art

[0002] As one of the major challenges affecting the healthy development of the global animal husbandry industry, parasitic nematode infection has long caused significant acute mortality and chronic wasting diseases in important economic animals such as cattle and sheep, seriously restricting the production efficiency and economic benefits of animal husbandry. Therefore, exploring and developing new, efficient and long-term parasitic nematode infection prevention and control technologies is of great significance to ensure the sustainable development of animal husbandry.

[0003] The life cycle of parasitic nematodes is complex, involving multiple key links such as worm development, infection establishment, parasitic stage transition, and complex interactions with the host. These biological processes are not only regulated by the genetic characteristics of the nematodes themselves, but also affected by host environmental factors, forming an intricate regulatory network. In-depth analysis of the molecular mechanisms of these key and specific biological processes is the premise and basis for screening potential vaccine targets and developing new prevention and control strategies.

[0004] In recent years, with the rapid development of molecular biology and genetics, scientists have gained a deeper understanding of the biological characteristics of parasitic nematodes. In particular, the insulin / insulin-like growth factors ignalling (IIS), as one of the key signal transduction pathways regulating parasite-host interactions and nematode development, has received widespread attention. The core of the IIS pathway is the insulin-like receptor (Insulin-like receptor), which can receive signals from the bioactive molecule insulin (Insulin, INS) and insulin-like peptides (Insulin-like peptides, ILPs), and then regulate a series of gene networks related to nematode chemical perception, fat metabolism, development, etc. These regulatory effects not only determine the nematode's ability to adapt to the external environment, but also profoundly affect its developmental process and parasitic strategy.

[0005] Studies in the model organism Caenorhabditis elegans have shown that abnormal regulation of the IIS pathway often leads to phenotypic changes such as developmental defects, metabolic abnormalities, and slow response to external stimuli in nematodes. These findings provide important clues for understanding the function of the IIS pathway in parasitic nematodes. However, compared with model organisms, the IIS pathway of parasitic nematodes may have more complex and unique regulatory mechanisms. For example, in the early stages of this study, it was found that the IIS pathway of Haemonchus contortus, as an important animal parasitic nematode, has a unique signaling molecule composition and regulatory pattern. Haemonchus contortus itself only expresses endogenous, antagonistic insulin-like peptides, and relies on exogenous (i.e., host-derived) agonistic insulin to exert its biological functions. This discovery reveals the close connection between the IIS pathway of parasitic nematodes and the host environment, and also provides new ideas for intervening in parasitic nematode infections. Summary of the invention

[0006] Based on the deficiencies in the prior art, the present invention provides a siRNA for intervening the sex ratio of the Haemonchus twister population and its application. In the study of exploring the function of the insulin-like polypeptide receptor of Haemonchus twister, the present invention determined that after the infective third-stage larvae incubated with a small interfering RNA primer mixture orally infect animals, they can significantly block the early infection and worm development of Haemonchus twister, significantly reduce the worm load in ruminants, significantly reduce the proportion of female adults, and significantly reduce the amount of eggs laid by the worm, thereby playing a role in blocking the spread of Haemonchus twister disease to a certain extent. The present invention has a high application value for controlling Haemonchus twister disease.

[0007] The present invention provides the use of an insulin-like polypeptide receptor or a gene encoding the same as a target in the preparation of a drug for intervening in the sex ratio of a Haemonchus contortus population.

[0008] On the other hand, the present invention also provides the use of insulin-like polypeptide receptor or its encoding gene as a target in the preparation of a drug for preventing and treating haemonchus disease.

[0009] Preferably, the application is to inhibit the activity of insulin-like polypeptide receptor or reduce the expression of insulin-like polypeptide receptor encoding gene, and the insulin-like polypeptide receptor is derived from Haemonchus contortus ( Haemonchus contortus ), insulin-like polypeptide receptor gene HCON_00066590-00001. Preferably, the amino acid sequence of the insulin-like polypeptide receptor is shown in SEQ ID No.1, and the sequence of the insulin-like polypeptide receptor gene HCON_00066590-00001 is shown in SEQ ID No.2.

[0010] The present invention also provides the use of the compound for inhibiting the activity of insulin-like polypeptide receptor or reducing the expression of insulin-like polypeptide receptor encoding gene in the preparation of medicine for preventing and treating haemonchus disease.

[0011] In some embodiments of the present invention, the compound is a DNA or RNA that blocks the expression or transcription of insulin-like polypeptide receptor;

[0012] Or the compound is a pharmaceutical preparation of an inhibitor of insulin-like polypeptide receptor or a pharmaceutically acceptable salt, solvate or hydrate thereof;

[0013] Alternatively, the compound is an antibody against insulin-like polypeptide receptor.

[0014] Preferably, the compound is siRNA for knocking out insulin-like polypeptide receptor encoding gene or a vector for expressing the siRNA, and the sequence of the siRNA is sense strand 5'-CGAUGAAAUUCUAGUGGAAUG-3' and / or antisense strand 5'-UUCCACUAGAAUUUCAUCGGU-3'.

[0015] The administration of the siRNA sequence can significantly reduce the transcription level of the insulin-like polypeptide receptor gene in the insect body.

[0016] In the embodiment of the present invention, the administration of the siRNA sequence can activate the expression of cytochrome P450, a key gene for steroid hormone metabolism in the insect body, 24h and 72h later. daf-9 Nuclear hormone receptor genes daf-12 Upregulated expression.

[0017] In the examples of the present invention, the administration of the siRNA sequence can significantly inhibit the development of infective third-stage larvae.

[0018] The present invention also provides a drug for preventing and treating haemonchus nematode disease, wherein the drug is one of the following:

[0019] (1) Antibodies against insulin-like peptide receptor;

[0020] (2) Pharmaceutical preparations of inhibitors of insulin-like polypeptide receptors or pharmaceutically acceptable salts, solvates or hydrates thereof;

[0021] (3) DNA or RNA that inhibits the expression or transcription of insulin-like polypeptide receptor.

[0022] Preferably, the drug is siRNA for knocking out insulin-like polypeptide receptor encoding gene or a vector for expressing the siRNA, and the sequence of the siRNA is sense strand 5'-CGAUGAAAUUCUAGUGGAAUG-3' and / or antisense strand 5'-UUCCACUAGAAUUUCAUCGGU-3'.

[0023] The present invention also provides a drug for intervening in the sex ratio of a population of Haemonchus contortus, wherein the drug is one of the following:

[0024] (1) Antibodies against insulin-like peptide receptor;

[0025] (2) Pharmaceutical preparations of inhibitors of insulin-like polypeptide receptors or pharmaceutically acceptable salts, solvates or hydrates thereof;

[0026] (3) DNA or RNA that inhibits the expression or transcription of insulin-like polypeptide receptor.

[0027] Preferably, the drug is siRNA for knocking out insulin-like polypeptide receptor encoding gene or a vector for expressing the siRNA, and the sequence of the siRNA is sense strand 5'-CGAUGAAAUUCUAGUGGAAUG-3' and / or antisense strand 5'-UUCCACUAGAAUUUCAUCGGU-3'.

[0028] The present invention also provides a siRNA for intervening in the sex ratio of a Haemonchus contortus population, wherein the sequence of the siRNA is a sense strand 5'-CGAUGAAAUUCUAGUGGAAUG-3' and / or an antisense strand 5'-UUCCACUAGAAUUUCAUCGGU-3'.

[0029] Beneficial effects of the present invention:

[0030] The in vivo experiment of the present invention on ruminant Hu sheep confirmed that the daily ovulation amount of the attacking group after application of the siRNA primer of the present invention was significantly reduced, the peak of ovulation was delayed by 4 days compared with the normal group, the insect load of adults (especially females) was significantly reduced, and a certain transmission blocking effect was played. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Agarose gel electrophoresis of PCR products, M is DNA marker, 0 is the amplification of the target gene coding region. The PCR results show a single band around 4000 bp. Sequencing shows that the full length of the HCON_00066590-00001 coding region is 4332 bp;

[0032] Figure 2 Analysis of target gene expression levels after administration of the interfering primer RNAi of the present invention; wherein **** indicates p<0.0001;

[0033] Figure 3 The effect of the interfering primer RNAi of the present invention on the in vitro development rate of larvae;

[0034] Figure 4The effect of the interfering primer RNAi of the present invention on the body length and width of larvae; wherein, ** indicates p<0.01, and ns indicates p>0.05;

[0035] Figure 5 The effect of the interfering primer RNAi of the present invention on the in vitro activity of larvae; wherein ns means p>0.05;

[0036] Figure 6 The present invention monitors the dynamic changes of egg discharge from 18 to 42 days after passive attack;

[0037] Figure 7 The load of abomasum adults was counted 42 days after passive attack of the present invention, and compared with the control group, the load of abomasum adults was significantly reduced; *** indicates p<0.001, and * indicates p<0.05;

[0038] Figure 8 The sex ratio of abomasum adults was statistically analyzed 42 days after passive attack. Compared with the control group, the proportion of female worms was significantly reduced; where **** indicates p<0.0001, and ns indicates p>0.05. DETAILED DESCRIPTION

[0039] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0040] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0041] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0042] Sources of experimental animals and worm strains: The 3- to 5-month-old Hu sheep used in this experiment were purchased from Huzhou Meimie Sheep Animal Husbandry Co., Ltd. They were in good health and had no parasitic infection after examination; the infected worm strain of Haemonchus contortus was from the laboratory of Professor Du Aifang and Researcher Ma Guangxu of Zhejiang University.

[0043] Example 1: Cloning of target gene HCON_00066590-00001 and design and synthesis of siRNA primers

[0044] 1.1 Extraction of total RNA from Haemonchus contortus (Trizol method) and reverse transcription

[0045] Trizol method to extract total RNA:

[0046] (1) Take the eggs and worm bodies of Haemonchus contortus at different developmental stages and put them into a glass grinding rod (soaked in 1‰ DEPC water for 48 h before use, sterilized by high pressure and dried). Add 1 mL of Trizol, grind thoroughly on ice, transfer to a 1.5 mL Eppendorf (EP) tube, and let stand at room temperature for 5 min.

[0047] (2) Add chloroform (1 / 5 the volume of Trizol), shake vigorously for 20 seconds, and let stand for 10 minutes;

[0048] (3) Centrifuge at 12,000 rpm for 15 min at 4°C, pipette the upper aqueous phase into a new 1.5 mL EP tube, add 0.5 mL of isopropanol, mix well with a pipette, and let stand at room temperature for 10 min.

[0049] (4) Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, add 1 mL of 75% ethanol (anhydrous ethanol: 1‰ DEPC water = 3:1), gently invert to mix, and wash the precipitate;

[0050] (5) Centrifuge at 8000 rpm for 5 min at 4°C, discard the supernatant, and dry for several minutes until the precipitate turns transparent.

[0051] (6) Add 11 μL of enzyme-free sterile water to dissolve.

[0052] Reverse transcription:

[0053] (1) Add 1 μL Oligo(dT)20, mix well, and place in a 65°C water bath for 5 min, then immediately place on ice;

[0054] (2) Prepare the reaction solution according to the system in Table 1;

[0055] Table 1

[0056]

[0057] (3) After the reverse transcription reaction solution is mixed, incubate it in a 30°C water bath for 10 min, 42°C water bath for 1.5 h, 85°C water bath for 10 min, and 4°C ice bath for 5 min. Centrifuge it immediately. The reverse transcription reaction solution is cDNA and store it in a freezer at -20°C.

[0058] 1.2 Amplification of HCON_00066590-00001 gene

[0059] Using the amino acid sequence of the Caenorhabditis elegans insulin receptor gene WBGene00000898 (NP_001299916.1) as a template, BLAST was performed in WormBase to obtain the homologous sequence HCON_00066590-00001, the sequence of which is shown in SEQ ID No.1.

[0060] (1) Using the cDNA of adult Haemonchus contortus as a template, specific primers were designed for PCR amplification. The PCR reaction system is shown in Table 2; the primers are as follows:

[0061] 5'-ATGATGATGAAACGACGAAAAGTTCCGG-3',

[0062] 5'-TCAAACCTCATCTTCGGCTTCAGAA-3';

[0063] Table 2

[0064]

[0065] The PCR reaction program was as follows: 95°C for 5 min; 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min, 30 cycles; 16°C for 1 h;

[0066] (2) The PCR reaction products were separated and recovered by 1% agarose gel electrophoresis, and the recovered gel was purified using a DNA gel recovery kit and ligated to the pMD19-T (simple) vector. The ligated products were then transferred into TOP10 competent cells. After identification by bacterial liquid PCR, the positive bacterial liquid was sent to Zhejiang Easyside Biotechnology Co., Ltd. for sequencing analysis.

[0067] 1.3 TA cloning, transformation and sequencing

[0068] 1.3.1 TA cloning

[0069] The purified PCR product was connected to the pMD19-T (simple) vector to obtain the PCR product-pMD19-T (simple) plasmid. The reaction system is shown in Table 3;

[0070] Table 3

[0071]

[0072] After the reaction solution was mixed, it was connected to a 16°C constant temperature metal bath for 3 h.

[0073] 1.3.2 Conversion

[0074] (1) Thaw the E. coli TOP10 competent cells stored at -80℃ on ice, add the PCR product-pMD19-T (simple) plasmid to the competent cells, mix well, and place on ice for 25 min;

[0075] (2) Heat shock at 42°C for 45 s, followed by ice bath for 2 min;

[0076] (3) Add 1 mL of LB liquid medium and incubate at 37°C in a constant temperature shaker at 190 rpm for 45 min.

[0077] (4) Centrifuge at 4500 rpm for 5 min, discard part of the supernatant, retain 100 μL of the culture medium, pipette to mix, take 30 μL and spread it on ampicillin-resistant LB solid plate culture medium, and incubate in a 37°C incubator for 16 h.

[0078] 1.3.3 PCR identification of positive single colonies

[0079] (1) Pick a single colony and place it in 1 mL of ampicillin-resistant LB liquid culture medium. Incubate at 37°C in a constant temperature shaker at 190 rpm for 6 h.

[0080] (2) Using the shaken cultured bacterial solution as a template, perform bacterial solution PCR with the following identification primers. The PCR reaction system is shown in Table 4.

[0081] 5'-ATGATGATGAAACGACGAAAAGTTCG-3',

[0082] 5'-TTTGATGGATCTTCTTCCATTCCCT-3';

[0083] Table 4

[0084]

[0085] The PCR reaction program was as follows: 95°C for 5 min; 95°C for 15 s, 60°C for 30 s, 72°C for 1 min, 25 cycles; 72°C for 10 min; 16°C for 1 h;

[0086] (3) The PCR reaction products were separated by 1% agarose gel electrophoresis. Samples with bands that met the target size were considered positive. Figure 1 Agarose gel electrophoresis of the PCR product showed a single band at around 4000 bp, which was sent to Zhejiang Easyside Biotechnology Co., Ltd. for sequencing and analysis.

[0087] 1.3.4 Sequencing

[0088] The positive bacterial solution was sent to Zhejiang Easyside Biotechnology Co., Ltd. for sequencing analysis. The full length of the HCON_00066590-00001 coding region was 4332 bp, and the sequence was shown in SEQ ID No. 2. The positive plasmid containing the correct sequence was frozen at -20°C for future use.

[0089] 1.4 siRNA primer design and synthesis

[0090] Based on the HCON_00066590-00001 gene sequence, siRNA was designed using the DSIR website (http: / / biodev.extra.cea.fr / DSIR / DSIR.html). At-lhcb1 The primers designed based on the gene sequence of (NM_129568.3) were used as irrelevant controls and sent to Hangzhou Youkang Biotechnology Co., Ltd. for synthesis. The primer sequences are as follows:

[0091] siRNA sequence of HCON_00066590-00001 gene sequence:

[0092] Sense strand 5'-CGAUGAAAUUCUAGUGGAAUG-3',

[0093] Antisense strand 5′-UUCCACUAGAAUUUCAUCGGU-3′;

[0094] At-lhcb1 siRNA sequence of gene sequence:

[0095] Sense strand 5'-GAGAAGUUGGAUACUUUGAAG-3',

[0096] Antisense strand 5′-UCAAAGUAUCCAACUUCUCUG-3′.

[0097] Example 2: Purification, surface sterilization and desheathing of Haemonchus contortus

[0098] 2.1 Purification of Haemonchus contortus larvae

[0099] (1) Collect the third-stage (L3) larvae in the fecal culture medium, repeatedly rinse the fecal culture medium with PBS, and collect the larvae;

[0100] (2) Connect a rubber tube to the bottom of the funnel and clamp the lower end of the rubber tube with a clamp. Fill the rubber tube and the bottom of the funnel with PBS. At the same time, fold a 600-mesh nylon sieve into a triangular cone and place it on the glass funnel.

[0101] (3) Use a disposable plastic pipette to add the washed mixture to a 600-mesh nylon sieve in portions. The total volume of the mixture should not exceed 1 / 3 of the volume of the funnel. Let it stand at room temperature for 2-6 hours to allow the more active larvae to drill through the nylon sieve and sink to the bottom of the rubber tube.

[0102] (4) Every 2 h, release the clamp and use a 1.5 mL EP tube to collect 1 mL of PBS containing larvae;

[0103] (5) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash with PBS;

[0104] (6) Repeat step (5) twice and concentrate the larvae in 100 μL PBS.

[0105] 2.2 Surface sterilization of Haemonchus contortus larvae

[0106] (1) Add 1 mL of nematode sterilization solution to the EP tube containing the purified larvae, mix thoroughly on a multi-purpose rotary shaker at the lowest speed, and sterilize for 5 min; centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0107] (2) Repeat step (1) three times, discard the nematode sterilization solution, add 1 mL of physiological saline (sterile), mix thoroughly, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0108] (3) Repeat step (2) three times to completely remove the nematode sterilization solution. The sterilized larvae are stored in 100 μL of saline (sterile) for later use.

[0109] 2.3 L3 larvae desheathing

[0110] (1) Add 98 μL of NaClO solution to 10 mL of insect liquid and perform desheathing at 190 rpm in a 37°C constant temperature shaker for 25 min.

[0111] (2) Wash with sterile saline for 5 times, centrifuge at 1000 g for 5 min each time, discard the supernatant, wash with NaClO, take 10 μL of the larvae solution and count under a stereomicroscope. Count the number of L3 (xL3) larvae after sheathing, and dilute with DMEM medium (containing 1‰ penicillin-streptomycin double antibody) to about 6-7 larvae / μL, ensuring that there are 300 xL3 larvae in each well of the 96-well plate (50 μL / well).

[0112] Example 3: siRNA immersion method interference and effect evaluation

[0113] 3.1 siRNA soaking method

[0114] The interference experiment was divided into three groups: a. DMEM medium containing only xL3 larvae (300 larvae / well) was added as the blank control group; b. At-lhcb1 Primer (1 μL / well) as an irrelevant control group; c. Add HCON_00066590-00001 siRNA primer as an interference group. Each group was set up with 12 replicates to ensure that each well of the 50 μL interference system contained at least 300 xL3 larvae.

[0115] The immersion interference was performed in a 96-well plate, and the culture system was shown in Table 5;

[0116] Table 5

[0117]

[0118] Culture in a 38°C, 10% CO2 incubator.

[0119] 3.2 Real-time fluorescence quantitative PCR experiment after RNAi

[0120] (1) Synthesize cDNA from samples collected from RNAi experiments according to the method in 1.1;

[0121] (2) Using the above cDNA as template, perform real-time fluorescence quantitative PCR experiment with the following qPCR primers;

[0122] 5'-CTGCAGCGTAATCGAGGGAT-3',

[0123] 5′-ACAAATGAGGGCGAGAGTCG-3′;

[0124] (3) The real-time fluorescence quantitative PCR reaction system is shown in Table 6;

[0125] Table 6

[0126]

[0127] Real-time fluorescence quantitative PCR reaction program: 95°C for 1 min; 95°C for 15 s, 60°C for 15 s, 72°C for 30 s, 40 cycles; 95°C for 10 s, 65°C for 5 s, and 95°C for 0.5 s.

[0128] The expression level of target gene after RNAi with interference primers is shown in Figure 2 As shown, the results showed that compared with the blank control group, the transcription level of HCON_00066590-00001 in the interference group was significantly decreased, indicating that the interference primers can successfully interfere with the transcription of this gene.

[0129] 3.3 Phenotypic analysis after RNAi

[0130] (1) Development rate: The mouth pouch of the third-stage larvae has not yet formed, and the larvae cannot feed at this time; the mouth pouch is fully developed at the fourth stage, and the larvae begin to feed. According to the morphological development of the mouth pouch, the number of L3 larvae and the fourth stage (L4) larvae after the disturbance is recorded, and the ratio of L4 larvae is calculated as the L3 larval development rate;

[0131] The results are as follows Figure 3 As shown, compared with the blank control group, the worm development rate in the interference group decreased significantly on the 3rd, 4th, 5th, 6th and 7th days of culture, indicating that the gene can promote the development of the infective larvae of Haemonchus contortus to the parasitic larvae stage.

[0132] (2) Body length and width: 10 larvae were randomly selected 2, 4, and 6 days after the disturbance and placed under an Olympus DP23 biological microscope for observation. The body length and width were measured using Image J software. The length from the tip of the head to the tip of the tail was taken as the body length, and the width at the widest point in the middle of the body was taken as the body width.

[0133] The results are as follows Figure 4 As shown in the figure, compared with the blank control group, the body length of the interference group was significantly shortened on the 2nd, 4th and 6th day of culture, while the body width of the interference group was significantly shortened on the 4th and 6th day of culture. This indicates that the role of this gene may be to make it grow faster, but there is a limit to the plateau period.

[0134] (3) Activity detection: The activity of larvae was measured every 24 h within 72 h after the disturbance using a Wmicrotrscker small animal activity analyzer.

[0135] The results are as follows Figure 5 As shown, compared with the blank control group, there was no significant difference in the activity of the worms in the interference group at 0, 24, 48, and 72 hours of culture, indicating that the interference primers would not cause abnormal worm activity.

[0136] Example 4: Animal infection and protection experiment after silencing of Haemonchus contortus HCON_00066590-00001

[0137] 4.1 Experimental grouping and attack

[0138] (1) Sixteen Hu sheep aged 3 to 5 months were randomly divided into 4 groups, with 4 sheep in each group. They were kept in different pens and the pen numbers and ear tags of the experimental sheep were recorded. The negative control group was not attacked with insects, the positive control group was attacked with untreated normal xL3, and the experimental group (target gene RNAi) and irrelevant control group were attacked with xL3 interfered with by immersion for 72 hours.

[0139] (2) Three days before and after the parasite attack (including the day of the attack), that is, for 7 consecutive days, dexamethasone inhibitor was injected into the hind limb muscles of all experimental sheep to produce immunosuppression; each sheep in the blank control group was orally infused with 10 mL of normal saline, and each sheep in the other groups was orally infected with 8,000 normal xL3 and interfered xL3.

[0140] 4.2 Observation of protection effect

[0141] (1) Monitoring of ovulation volume and ovulation peak: The daily ovulation volume of Hu sheep was monitored by measuring the number of eggs per gram of feces, and the peak time of ovulation was analyzed based on the changing trend of the daily ovulation volume. Fresh feces of each experimental sheep was collected every day from 18 days after infection until before slaughter, and the eggs were counted under a microscope using a McMaster egg counting plate. The group names and ear tags of each sheep were carefully recorded and corresponded to the data results.

[0142] result( Figure 6 ) showed that no nematode eggs were detected in the feces of the hosts in the blank control group. The eggs in the host feces of the target gene RNAi group appeared later than those in the positive infection group, the ovulation volume was significantly reduced, and the peak of ovulation was delayed by 4 days.

[0143] (2) 42 days after the challenge, all the experimental sheep were euthanized, the abomasum was removed, and the number of adult Haemonchus contortus colonized in the abomasum of each group of experimental sheep was counted, which was the worm load.

[0144] result( Figure 7 ) showed that the target gene RNAi group could significantly reduce the adult worm load in Hu sheep compared with the positive control group.

[0145] (3) Calculate the sex ratio according to the following formula: Female sex ratio = number of female insect loads / number of male insect loads.

[0146] result( Figure 8 ) showed that the target gene RNAi group could significantly reduce the proportion of female worms in Hu sheep compared with the positive control group.

[0147] The above is only the embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Use of a compound for inhibiting the activity of insulin-like polypeptide receptor or reducing the expression of insulin-like polypeptide receptor encoding gene in the preparation of a drug for preventing and treating haemonchus nematode disease; The compound is a siRNA for knocking out an insulin-like polypeptide receptor coding gene or a vector for expressing the siRNA. The sequence of the siRNA is a sense chain 5'-CGAUGAAAUUCUAGUGGAAUG-3' and an antisense chain 5'-UUCCACUAGAAUUUCAUCGGU-3'.

2. A drug for preventing and treating haemonchus nematode disease, characterized in that: The drug is siRNA for knocking out insulin-like polypeptide receptor coding gene or a vector for expressing the siRNA, and the sequence of the siRNA is a sense chain 5'-CGAUGAAAUUCUAGUGGAAUG-3' and an antisense chain 5'-UUCCACUAGAAUUUCAUCGGU-3'.

3. A drug for intervening in the sex ratio of a population of Haemonchus contortus, characterized in that: The drug is siRNA for knocking out insulin-like polypeptide receptor coding gene or a vector for expressing the siRNA, and the sequence of the siRNA is a sense chain 5'-CGAUGAAAUUCUAGUGGAAUG-3' and an antisense chain 5'-UUCCACUAGAAUUUCAUCGGU-3'.

4. An siRNA for intervening the sex ratio of a Haemonchus contortus population, characterized in that: The sequences of the siRNA are: sense strand 5'-CGAUGAAAUUCUAGUGGAAUG-3' and antisense strand 5'-UUCCACUAGAAUUUCAUCGGU-3'.

Images