Double-stranded RNA for killing schistosoma japonicum and its application

Abstract

The present invention relates to the field of molecular biology and biomedical technology, and in particular to a double-stranded RNA for killing schistosomes and its application. The double-stranded RNA is composed of a sense strand shown in SEQ ID NO.1 and an antisense strand shown in SEQ ID NO.2. Through animal in vivo experiments, it is confirmed that the double-stranded RNA can produce a significant killing effect on the worm body after schistosome infection. This double-stranded RNA can prevent schistosomes from establishing infection in the host body and inhibit the growth and development of the worm body. At the same time, it can also significantly reduce the egg-laying rate of female worms and kill the worm body that has established infection, thereby helping to reduce the host pathological damage caused by schistosome infection. Therefore, this double-stranded RNA is a highly potential schistosomiasis therapeutic agent, suitable for the treatment after schistosome infection and can provide new ideas for vaccine research and development.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and biomedicine technology, and in particular to a double-stranded RNA for killing schistosomes and an application thereof. Background Art

[0002] Schistosomiasis is a zoonotic parasitic disease that seriously endangers human public health. It is caused by schistosomes. Currently, more than 230 million people are infected with schistosomes worldwide, resulting in about 280,000 deaths each year. There are three main types of schistosomes known to infect humans: Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium. Among them, Schistosoma japonicum is famous for its amazing egg-laying speed and egg-laying volume. A large number of eggs produced by the worm enter the host tissues and organs through the blood circulation and are deposited there, causing inflammation and fibrosis reactions in the host organs, leading to severe pathological damage and even the death of the patient.

[0003] At present, the treatment of schistosomiasis relies on a single drug, praziquantel, which is a widely used antiparasitic drug with high efficacy against a variety of parasitic infections. Its main mechanism of action is to increase the permeability of the parasite cell membrane, leading to the loss of intracellular calcium ions, thereby causing paralysis and death of the parasite muscles. It has the characteristics of few side effects and high efficacy. However, with the long-term and large-scale use of praziquantel, drug resistance has emerged in some areas. In addition, praziquantel is unable to act on larvae and cannot effectively resist secondary infections. Therefore, finding new drug targets against schistosomiasis and developing new treatments have become public health issues that need to be urgently addressed.

[0004] Schistosoma has a very complex life cycle, mainly going through six developmental stages: eggs, miracidia, sporocysts, cercariae, juveniles, and adults, and two parasitic stages in the intermediate host snail and the definitive host mammal. The life history of schistosomes is very complex, and there is a lack of immortalized cell lines that can be cultured for a long time, which poses a huge challenge to gene editing and gene function research. These factors have jointly led to the lack of schistosome gene editing technology, which in turn has hindered the development of anti-schistosomal drugs or vaccines.

[0005] Currently, only RNA interference is widely used in the study of schistosome gene function. Its principle is to regulate gene expression through specific short interfering RNA. When double-stranded RNA is cut into siRNA by Dicer enzyme, one strand of siRNA is absorbed by RNA-induced silencing complex to form an active complex, which recognizes and binds to target mRNA through complementary pairing, causing mRNA to be cut and degraded, thereby specifically inhibiting the expression of specific genes. Therefore, it is necessary to determine which specific genes can be inhibited to reduce the worm load and reduce the pathological damage of schistosome infection to the host, so as to further expand the application to the functional study of genes related to worm parasitism and reproductive development in the host, and provide new treatment methods for anti-schistosome. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention aims to provide a double-stranded RNA for killing schistosomes and its application. The double-stranded RNA can significantly block the growth and development of schistosomes and kill the schistosomes in the body during the establishment stage of schistosome infection; in addition, in the mature worm stage after schistosome infection, inhibiting the expression of this gene can also effectively interfere with the normal reproduction of the worm, inhibit female worms from laying eggs, reduce the degree of liver granulomas, and thus reduce pathological damage to the host.

[0007] Therefore, one of the objects of the present invention is to provide a double-stranded RNA for killing schistosomes, which consists of a sense strand shown in SEQ ID NO.1 and an antisense strand shown in SEQ ID NO.2.

[0008] The second object of the present invention is to provide the use of the double-stranded RNA for killing schistosoma in the preparation of anti-schistosoma drugs.

[0009] Furthermore, the schistosome is Schistosoma japonicum.

[0010] Furthermore, the drug is used for:

[0011] 1) Block the growth and development of Schistosoma japonicum in the host;

[0012] 2) Killing Schistosoma japonicum in the host;

[0013] 3) Interfere with the reproduction of Schistosoma japonicum in the host;

[0014] 4) Reduce the pathological damage of Schistosoma japonicum to the host liver in the host body.

[0015] Furthermore, 1) and 2) act on the host 1d-30d after Schistosoma japonicum infects the host.

[0016] Furthermore, 3) it acts on the host 26d-42d after Schistosoma japonicum infects the host.

[0017] Furthermore, interfering with the reproduction of Schistosoma japonicum includes: inhibiting the development of reproductive organs of Schistosoma japonicum and inhibiting the egg laying of Schistosoma japonicum.

[0018] Furthermore, reducing the pathological damage of Schistosoma japonicum to the host liver includes: reducing the number of liver granulomas, reducing the area of ​​liver granulomas, and reducing the degree of liver inflammatory cell infiltration and fibrosis.

[0019] Furthermore, the drug also includes one or more of a pharmaceutically acceptable carrier, diluent or excipient.

[0020] Furthermore, the dosage form of the drug is injection, oral preparation or spray, and the oral preparation is suspension, capsule, tablet, pill or granule.

[0021] Beneficial effects:

[0022] The present invention provides a double-stranded RNA for killing schistosomes, establishes its therapeutic dosage form, and explores its usage and dosage. Through animal experiments, it is found that the double-stranded RNA has a good killing effect on schistosomes in the early and mature stages after infection with schistosomes, can not only prevent schistosomes from establishing infection in the host and effectively inhibit the growth and development of the schistosomes, but also can reduce the amount of eggs laid by female schistosomes and kill the schistosomes that have established infection, ultimately reducing the pathological damage to the host. The double-stranded RNA has great application value, can be used to treat schistosome infections at different developmental stages, and can effectively treat schistosomiasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A is a schematic diagram of the usage and dosage of double-stranded RNA in the early stage of schistosomiasis infection establishment and in the mature worm stage after schistosomiasis infection is established; B is a schematic diagram of the usage and dosage of double-stranded RNA in the mature worm stage after schistosomiasis infection is established.

[0024] Figure 2 Schematic diagram of the statistical results of the recovery rate and worm length of Schistosoma japonicum after injection of dsRNABip / dsRNAGFP in the early stage of Schistosoma infection establishment and the mature worm stage after the infection is established; A is a comparison of the number of Schistosoma japonicum worms recovered after injection of dsRNABip / dsRNAGFP in the early stage of Schistosoma infection establishment; B is a comparison of the length of Schistosoma japonicum worms after injection of dsRNA Bip / dsRNA GFP in the early stage of Schistosoma infection establishment; C is a comparison of the number of Schistosoma japonicum worms recovered after injection of dsRNA Bip / dsRNA GFP in the mature worm stage after the infection is established; D is a comparison of the length of Schistosoma japonicum worms after injection of dsRNA Bip / dsRNA GFP in the mature worm stage after the infection is established.

[0025] Figure 3 Schematic diagram of the comparison of Carmine staining and Fastblue B staining of the reproductive organs of Schistosoma japonicum after injection of dsRNA Bip and dsRNA GFP in the mature worm stage after infection is established; A is a comparison of Carmine staining of the reproductive organs of Schistosoma japonicum, and B is a comparison of Fastblue B staining of the yolk gland of Schistosoma japonicum.

[0026] Figure 4 These are upright laser confocal observations of the reproductive organs of the worms after injection of dsRNA Bip and dsRNA GFP at the mature worm stage after infection is established.

[0027] Figure 5 Schematic diagram comparing the spleen and liver of mice after injection of dsRNA Bip and dsRNA GFP at the mature worm stage after infection is established; A is the spleen and B is the liver.

[0028] Figure 6 These are the results of detecting the number of worm eggs in the liver after injection of dsRNA Bip and dsRNA GFP at the mature worm stage after infection is established, as well as the results of HE staining and Masson staining of the liver. A is the result of detecting the number of worm eggs in the liver, and B is the result of HE staining and Masson staining of the liver.

[0029] Figure 1-Figure 6 The abbreviations dsBip and dsGFP in the table all indicate the injection of dsRNA Bip, and dsGFP all indicate the injection of dsRNA GFP. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] Example 1. Preparation of double-stranded RNA

[0032] 1. Synthesis of DNA template

[0033] (1) Amplification of the target gene SjBip

[0034] Primer primer 6 was used to design dsRNA primers for the SjBip gene of Schistosoma japonicum, and cDNA of Schistosoma japonicum was used as a template to amplify using the dsRNA primers.

[0035] The SjBip gene is referenced to NCBI database AF044412.1, and the nucleotide sequence is shown in SEQ ID NO.3.

[0036] The dsRNA primers are shown in SEQ ID NO.4 and SEQ ID NO.5:

[0037] SEQ ID NO.4: 5′-taatacgactcactatagggagaTGGTGTCAAGTGAAGATAAG-3′, the sequence shown in lowercase letters is the T7 promoter sequence.

[0038] SEQ ID NO.5: 5′-taatacgactcactatagggagaGTTGGTTCGTTAATGATTCG-3′, the sequence shown in lowercase letters is the T7 promoter sequence.

[0039] PCR amplification program: the reaction program is 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 40s, 37 cycles; 72℃ final extension for 10min; 16℃ temporary storage.

[0040] Gel recovery: After amplification, cut a single target band under a UV gel irradiator, place the gel strip in a 1.5mL centrifuge tube, weigh the gel strip, add sol solution at a ratio of 1000μL / g, place the centrifuge tube in a 60℃ metal bath until the gel block in the tube is completely dissolved, add the gel solution to the DNA recovery column, let it stand for 1min, centrifuge at 12000r / min for 1min, and discard the waste liquid; add 700μL of rinse solution to the recovery column, gently shake the centrifuge tube and centrifuge at 12000r / min for 1min, and discard the waste liquid; centrifuge at 12000r / min for 2min with an empty column, discard the waste liquid, replace the collection tube and dry it at room temperature for 10min; after drying, add the elution solution preheated at 60℃ to the recovery column, centrifuge at 12000r / min for 2min, collect the DNA solution and store it at -20℃ for later use.

[0041] The nucleotide sequence of the amplified fragment of the SjBIP gene is shown in SEQ ID NO.6.

[0042] (2) Amplification of the control gene GFP

[0043] Primers were designed based on the sequence of the green fluorescent protein GFP vector plasmid constructed in the laboratory. The primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8:

[0044] SEQ ID NO. 7: 5'-TAATACGACTCACTATAGGGAGAAGTCAGTGGAGA GGGTGAAG-3'.

[0045] SEQ ID NO. 8: 5'-TAATACGACTCACTATAGGGAGAACTAGTTCTACGG ATCCATC-3'.

[0046] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, 37 cycles; final extension at 72°C for 10 min; and temporary storage at 16°C.

[0047] Gel recovery: After amplification, cut a single target band under a UV gel irradiator, place the gel strip in a 1.5mL centrifuge tube, weigh the gel strip, add sol solution at a ratio of 1000μL / g, place the centrifuge tube in a 60℃ metal bath until the gel block in the tube is completely dissolved, add the gel solution to the DNA recovery column, let it stand for 1min, centrifuge at 12000r / min for 1min, and discard the waste liquid; add 700μL of rinse solution to the recovery column, gently shake the centrifuge tube and centrifuge at 12000r / min for 1min, and discard the waste liquid; centrifuge at 12000r / min for 2min with an empty column, discard the waste liquid, replace the collection tube and dry it at room temperature for 10min; after drying, add the elution solution preheated at 60℃ to the recovery column, centrifuge at 12000r / min for 2min, collect the DNA solution and store it at -20℃ for later use.

[0048] The nucleotide sequence of the GFP gene amplified fragment is shown in SEQ ID NO.9.

[0049] 2. Synthesis of double-stranded RNA

[0050] The reaction system for double-stranded RNA, i.e., dsRNA synthesis, is as follows:

[0051] Table 1 Reaction system for dsRNA synthesis

[0052] Element Addition amount T7 reaction buffer 2μL T7 enzyme mix 2μL ATP solution 2μL UTP solution 2μL GTP solution 2μL CTP solution 2μL DNA template 200ng DEPC treated water Make up to 20 μL

[0053] The above system was added into a 1.5 mL centrifuge tube, shaken and mixed, and then incubated at 37°C overnight.

[0054] 3. Purification of double-stranded RNA

[0055] 1) The product of the above 37°C overnight incubation was centrifuged and placed in a 75°C water bath for 5 min to terminate the reaction;

[0056] 2) Add 1 μL TURBO DNase to the reaction system, shake thoroughly to mix, and place in a 37°C water bath for 15 min to remove the DNA template;

[0057] 3) Add 115 μL Nuclease-free water to the above system and 15 μL AmmoniumAcetate Stop Solution, and shake to mix;

[0058] 4) Add 50 μL of isopropanol, shake and mix, and place at -20°C for precipitation for at least 1 hour;

[0059] 5) After taking out the centrifuge tube, centrifuge at 13000rpm and 4℃ for 15min. After centrifugation, transparent precipitate can be observed at the bottom of the tube. During this process, transparent crystals can be observed.

[0060] 6) Use a pipette to remove the supernatant, add 700 μL 75% ethanol to wash the precipitate, and centrifuge at 13000 rpm, 4°C for 5 min;

[0061] 7) Use a pipette to discard the residual ethanol, open the lid and let it air-dry at room temperature for 15 minutes, and proceed to the next step after the ethanol has fully evaporated;

[0062] 8) Add 40 μL of Nuclease-free water to dissolve the precipitate, shake and mix, and denature in a 65°C water bath for 15 min;

[0063] 9) Detect dsRNA concentration using NanoDrop 2000;

[0064] 10) PCR detection to see if the size of the synthesized dsRNA fragment is consistent with the target gene. The loading volume is 0.2 μL of synthetic product, 1 μL of 10× loading buffer, and 8.8 μL of DEPC water;

[0065] 11) After confirming that the band size is correct, the two synthesized dsRNAs are named dsRNA Bip and dsRNA GFP, respectively, and stored at -80°C for future use.

[0066] 12) The nucleotide sequence of the sense strand of the synthesized dsRNABip is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2.

[0067] 13) The nucleotide sequence of the sense strand of the synthesized dsRNA GFP is shown in SEQ ID NO.10, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.11.

[0068] Example 2: In vivo interference experiment of double-stranded RNA

[0069] 1. In vivo interference of double-stranded RNA during the early stage of Schistosoma japonicum infection

[0070] Kunming mice were percutaneously infected with 60 cercariae and randomly divided into groups of 3 mice each. 20 μg / mouse of the target gene dsRNA Bip diluted with saline was injected through the tail vein on the 1st, 6th, 10th, 14th, 18th, 22nd, and 26th day after infection, for a total of 7 injections. The control group was injected with an equal amount of dsRNA GFP of the irrelevant interference fragment GFP. The parasites were obtained by perfusion method on the 30th day after infection ( Figure 1 (A).

[0071] 2. In vivo interference of double-stranded RNA in mature worm stage after schistosoma infection is established

[0072] Kunming mice were percutaneously infected with 40 cercariae and randomly divided into groups of 3 mice each. On the 26th, 30th, 34th, and 38th days after infection, 20 μg / mouse of the target gene dsRNA Bip was injected through the tail vein, diluted with saline, for a total of 4 injections. The control group was injected with an equal amount of dsRNA GFP of the irrelevant interference fragment. On the 42nd day after infection, the parasites were obtained by perfusion method ( Figure 1 (middle B).

[0073] 3. Effect of double-stranded RNA interference experiment in vivo

[0074] (1) Evaluation of treatment effect

[0075] 1) Evaluation of insect recovery rate and insect growth and development

[0076] After 30 / 42 days of schistosome infection, schistosomes were obtained from mice by perfusion and placed in a cell culture dish. 0.25% anesthetic prepared in DMEM medium containing 10% FBS was added to the cell culture dish. The cell culture dish was placed on a shaker until the male and female worms undid their clasped arms and separated completely. The worm bodies were collected in a centrifuge tube and fixed overnight with AFA solution. On the next day, the worm bodies were placed on a ruler slide to measure the worm length and count the number of recovered worm bodies. ImageJ software was used to measure and convert the worm body length.

[0077] 2) Carmine staining of insect bodies

[0078] Kunming mice were percutaneously infected with 40 cercariae and randomly divided into groups of 3 mice each. 20 μg / mouse of the target gene dsRNA Bip was injected through the tail vein at 26, 30, 34, and 38 days after infection, diluted with saline, for a total of 4 injections. The control group was injected with an equal amount of dsRNA GFP of the irrelevant interference fragment GFP. Schistosoma was obtained on the 42nd day after infection, and AFA solution was added for overnight fixation and then used for carmine staining. The specific staining steps are as follows:

[0079] ① Move 10-20 AFA-fixed worms to distilled water and soak overnight;

[0080] ② Move the rehydrated male and female worms to a 6-well cell culture plate, treat with glacial acetic acid for 15 minutes, discard the glacial acetic acid, and soak in distilled water for 1 hour;

[0081] ③Discard the distilled water and add 2.5mL of 35%, 50%, and 70% ethanol in turn for dehydration, each time for 15 minutes;

[0082] ④Discard 70% ethanol, add 2.5mL alum carmine staining solution, and stain for 2 minutes;

[0083] ⑤Discard the staining solution, add 2.5mL of alum carmine color separation solution, and soak for 30 minutes;

[0084] ⑥Discard the separation solution and add 35%, 50%, and 70% ethanol in sequence to gradually dehydrate, each time for 15 minutes;

[0085] ⑦ Discard 70% ethanol and add 2% hydrochloric acid alcohol decolorizing solution, changing the decolorizing solution several times until the insect body turns pink. When the insect body turns pink, add 70% ethanol to stop the decolorization;

[0086] ⑧Add 85%, 95%, and 100% ethanol in sequence to continue gradient dehydration, each time for 15 minutes. After dehydration is completed, transfer the insect body to methyl salicylate and soak for 5 minutes;

[0087] ⑨ Use gum to seal the transparent insect body, and try to avoid creating bubbles during the process;

[0088] ⑩ After the gum solidifies, place the specimen containing the insect body under a fluorescence microscope and a Nikon upright laser confocal microscope for observation. The excitation wavelength of the confocal microscope is 488nm.

[0089] 3) Detection of the number of eggs laid by insects

[0090] Kunming mice were percutaneously infected with 40 cercariae and randomly divided into groups of 3 mice each. 20 μg / mouse of the target gene dsRNA Bip was injected through the tail vein on days 26, 30, 34, and 38 after infection, diluted with saline, for a total of 4 injections. The control group was injected with an equal amount of dsRNA GFP of the irrelevant interference fragment GFP. The livers of mice were obtained on day 42 after infection, and the number of eggs and hatching rate in the livers were detected.

[0091] Among them, the statistics of the number of insect eggs are:

[0092] The right lobe of the liver of a mouse infected with Schistosoma japonicum for 42 days was placed in a 50 mL centrifuge tube and the liver weight was measured.

[0093] ① Add 20 mL of 5% NaOH solution and digest overnight at room temperature;

[0094] ② Vortex until no large liver tissue is visible;

[0095] ③ Centrifuge at 5000 rpm for 10 min and discard the supernatant;

[0096] ④ Resuspend the precipitate with 1.2% NaCl and adjust the volume to 10 mL;

[0097] ⑤ Take 10 μL of suspension slides and count the number of eggs. Do 10 counts for each sample;

[0098] ⑥ Calculate the number of eggs per gram of liver: number of eggs per gram of liver = average value of 10 egg statistics × 100 × 10 / liver weight.

[0099] (2) In vivo interference effects during the early stage of schistosoma japonicum infection establishment

[0100] The experimental results are as follows Figure 2 The results showed that the number of insects obtained on the 30th day was only 2 ± 0.5774 in the experimental group, and 38.33 ± 0.8819 in the control group, with a reduction rate of 94.73% ( Figure 2 The development of the insects in the experimental group was severely blocked. The average length of male insects in the interference group was 2.77±0.42mm, and that in the control group was 10.24±0.24mm. The average length of female insects in the interference group was 3.03±1.21mm, and that in the control group was 12.36±0.30mm ( Figure 2 (middle B).

[0101] (3) In vivo interference effects at the mature worm stage after schistosoma infection is established

[0102] The experimental results are as follows Figure 2 As shown: SjBip expression was inhibited on the 26th day, and the results of obtaining worms on the 42nd day showed that the worm reduction rate was 87.50% ( Figure 2 Middle C); males in the interference group shortened by 2.77±0.64mm, and females shortened by 7.85±0.72mm ( Figure 2 (middle D).

[0103] (4) Effects on the development of reproductive organs of insects

[0104] The experimental results are as follows Figure 3 As shown: Alum carmine staining results show that the ovaries of the female insects in the interference group are degenerated and shrunken, there are no mature yolk gland cells in the yolk gland, the transverse diameter is significantly reduced, and the testicles of the male insects are degenerated and shrunken, and the volume is significantly reduced ( Figure 3 Fastblue B staining results showed that the lipid droplet accumulation and pigment deposition in the yolk gland of female worms in the interference group were significantly reduced, accompanied by yolk gland degeneration ( Figure 3Confocal laser scanning microscopy showed that after SjBip was interfered, the number of mature oocytes in the ovaries of the worms decreased, the volume of yolk gland cells decreased, and they no longer presented a regular bundle structure. There were fewer mature spermatocytes in the testes, and the proportion of immature spermatocytes increased ( Figure 4 ).

[0105] (5) Impact on host pathological damage

[0106] The experimental results are as follows Figure 5 As shown: Compared with the control group, the spleen of mice did not swell after interfering with SjBip expression, but remained normal in size, indicating that inhibiting SjBip expression can reduce the intensity of immune response in the spleen of mice ( Figure 5 In addition, the number and distribution area of ​​liver granulomas in the SjBip interference group were significantly reduced compared with those in the control group ( Figure 5 Middle B), the experimental results in this part confirmed that inhibiting the expression of SjBip can reduce the pathological damage of schistosomes to the host.

[0107] (6) Effect on insect egg laying

[0108] According to statistics, the number of worm eggs in the liver of mice in the SjBip interference group was significantly lower than that in the control group. The number of worm eggs per gram of liver in the experimental group was 19072±3437, while that in the control group was 47846±7451, and the egg reduction rate was 60.14% ( Figure 6 (A). This indicates that the injection of dsRNABip inhibited the egg laying of Schistosoma japonicum. At the same time, the results of HE and Masson staining of liver tissue showed that the degree of eosinophilic granulomatous lesions, inflammatory cell infiltration and fibrosis in the liver of mice in the control group was more obvious than that in the experimental group ( Figure 6 Middle B), the experimental results of this part confirmed that inhibiting the expression of SjBip can reduce the pathological damage of worm eggs to the host.

[0109] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0110] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A double-stranded RNA for killing schistosoma japonicum, characterized in that: It consists of the sense strand shown in SEQ ID NO.1 and the antisense strand shown in SEQ ID NO.

2.

2. Use of the double-stranded RNA for killing schistosoma according to claim 1 in the preparation of anti-schistosoma drugs.

3. The use of the double-stranded RNA for killing schistosoma according to claim 2 in the preparation of an anti-schistosoma drug, characterized in that: The schistosoma is Schistosoma japonicum.

4. The use of the double-stranded RNA for killing schistosoma according to claim 3 in the preparation of an anti-schistosoma drug, characterized in that: The drug is used to: 1) Block the growth and development of Schistosoma japonicum in the host; 2) Killing Schistosoma japonicum in the host; 3) Block the reproduction of Schistosoma japonicum in the host; 4) Reduce the pathological damage of Schistosoma japonicum to the host liver in the host body.

5. The use of the double-stranded RNA for killing schistosoma according to claim 4 in the preparation of an anti-schistosoma drug, characterized in that: 1) and 2) act on the host 1d-30d after Schistosoma japonicum infects the host.

6. The use of the double-stranded RNA for killing schistosoma according to claim 4 in the preparation of an anti-schistosoma drug, characterized in that: 3) It acts on the host 26 days to 42 days after Schistosoma japonicum infects the host.

7. The use of the double-stranded RNA for killing schistosoma according to claim 4 in the preparation of an anti-schistosoma drug, characterized in that: Blocking the reproduction of Schistosoma japonicum includes: inhibiting the development of reproductive organs and egg laying of Schistosoma japonicum.

8. The use of the double-stranded RNA for killing schistosoma japonicum in the preparation of an anti-schistosoma japonicum drug according to claim 4, characterized in that: Reducing the pathological damage of Schistosoma japonicum to the host liver includes: reducing the number of liver granulomas, reducing the area of ​​liver granulomas, and reducing the degree of liver inflammatory cell infiltration and fibrosis.

9. The use of the double-stranded RNA for killing schistosoma japonicum in the preparation of an anti-schistosoma japonicum drug according to claim 2, characterized in that: The drug also includes one or more of a pharmaceutically acceptable carrier, a diluent or an excipient.

10. The use of the double-stranded RNA for killing schistosoma according to claim 2 in the preparation of an anti-schistosoma drug, characterized in that: The dosage form of the drug is injection, oral preparation or spray, and the oral preparation is suspension, capsule, tablet, pill or granule.

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