DsRNA of targeted IPV NS1 gene and application of dsRNA

Abstract

The invention discloses dsRNA (double-stranded ribonucleic acid) of a targeted IPV (infectious precocity virus) NS1 gene and application of the dsRNA. The dsRNA disclosed by the invention consists of a positive-sense strand of which the nucleotide sequence is as shown in SEQ ID NO.1 and an antisense strand of which the nucleotide sequence is as shown in SEQ ID NO.2. The dsRNA is injected into the macrobrachium rosenbergii infected with the IPV virus, so that the load of the IPV virus can be obviously reduced by utilizing the dsRNA, and the growth inhibition of the IPV virus on the macrobrachium rosenbergii is relieved. On the basis, the dsRNA or a biological material capable of expressing the dsRNA, such as a cell or a strain capable of expressing the dsRNA, can be used for preparing a reagent for inhibiting the IPV virus, so that viral diseases caused by IPV virus infection are prevented and treated. The composition is beneficial to prevention and treatment of IPV virus related diseases, and promotes the long and stable development of the macrobrachium rosenbergii breeding industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering. More specifically, it relates to a dsRNA targeting the NS1 gene of IPV virus and its application. Background Art

[0002] Macrobrachium rosenbergii is an important main paddy cultured shrimp variety. In recent years, during the cultivation of Macrobrachium rosenbergii, there has been a phenomenon that the shrimps cannot grow large, which is called "iron prawn syndrome" (IPS) by farmers. The "iron prawn syndrome" was discovered in 2010 and shows a spreading trend. It has now become a common and easily-occurring disease of Macrobrachium rosenbergii, bringing a lot of losses to farmers and being one of the most important threats to the Macrobrachium rosenbergii aquaculture industry.

[0003] With the development and wide application of metagenomics technology, researchers have discovered a new virus belonging to the family Flaviviridae from precocious Macrobrachium rosenbergii, named Infectious precocity virus (IPV), which is the pathogen of "iron prawn syndrome". The typical symptoms of "iron prawn syndrome" caused by IPV infection in Macrobrachium rosenbergii are: individual growth retardation, miniaturization, and hardening of the shrimp shell; when the shrimp grows to 5 - 6 cm, the second appendage segment becomes significantly wider, the villi at the end of the cheliped turn blue and become rigid, similar to the characteristics of sexual maturity. Although Macrobrachium rosenbergii infected with IPV will not show large-scale death, its yield will be significantly reduced, less than 50% of that of healthy shrimps, and at the same time, the feed conversion ratio is relatively high, seriously affecting the economic benefits. Currently, the research on IPV virus mainly focuses on virus detection and epidemiological investigation, etc. There is no preparation or method for specifically targeting IPV to inhibit it and prevent and control "iron prawn syndrome".

[0004] Double-stranded RNA (dsRNA) is a molecule composed of two complementary RNA strands. RNA interference mediated by it can be used to inhibit virus replication. Although there have been reports on dsRNA that can inhibit flavivirus, the genes of different viruses in the same family are not the same. Even if there are homologous genes, the functions of the genes are not necessarily the same. In addition, even different RNAi molecules designed for the same target may have completely different functions. For IPV virus, the functions of its various genes have not been clearly defined yet, and it is also not clear how to obtain dsRNA that can significantly inhibit the proliferation of IPV virus and effectively relieve the growth inhibition of IPV on Macrobrachium rosenbergii. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides a dsRNA targeting the NS1 gene of IPV virus (Infectious precocity virus) and its application. The replication of IPV virus can be effectively inhibited by using the dsRNA.

[0006] The first object of the present invention is to provide a dsRNA targeting the NS1 gene of the infectious precocious puberty virus.

[0007] The second object of the present invention is to provide a nucleic acid molecule encoding the dsRNA.

[0008] The third object of the present invention is to provide an expression cassette containing the nucleic acid molecule.

[0009] The fourth object of the present invention is to provide a vector containing the nucleic acid molecule or the expression cassette.

[0010] The fifth object of the present invention is to provide a cell capable of expressing the dsRNA.

[0011] The sixth object of the present invention is to provide a strain capable of expressing the dsRNA.

[0012] The seventh object of the present invention is to provide a reagent containing the dsRNA, the nucleic acid molecule, the expression cassette, the vector, the cell and / or the strain.

[0013] The eighth object of the present invention is to provide the use of the dsRNA, the nucleic acid molecule, the expression cassette, the vector, the cell, the strain or the reagent in the preparation of a product for inhibiting the infectious precocious puberty virus.

[0014] The ninth object of the present invention is to provide the use of the dsRNA, the nucleic acid molecule, the expression cassette, the vector, the cell, the strain or the reagent in the preparation of a product for preventing and treating diseases caused by infectious precocious puberty virus infection.

[0015] The above objects of the present invention are achieved by the following technical solutions:

[0016] Based on the genome of the IPV virus, the present invention designed and synthesized a plurality of different dsRNAs, and obtained a dsRNA that can effectively inhibit the replication of the IPV virus. Therefore, the present invention claims protection for the dsRNA and its applications.

[0017] Specifically, the dsRNA targets the NS1 gene of the IPV virus, that is, the present invention provides a dsRNA targeting the NS1 gene of the IPV virus, and the dsRNA is composed of a sense strand with a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.2; the DNA sequence of the NS1 gene is as shown in SEQ ID NO.3.

[0018] The present invention also provides a method for obtaining the dsRNA.

[0019] As an alternative embodiment, the obtaining method is: directly synthesizing the dsRNA by chemical synthesis method.

[0020] As an alternative embodiment, the obtaining method is: using in vitro transcription technology, transcribing the cDNA template of IPV virus into RNA by T7 phage RNA polymerase (T7 RNAP), and then enabling two complementary RNA strands to renature to form dsRNA through specific steps.

[0021] In a specific embodiment of the present invention, the primers for synthesizing the dsRNA include NS1-F (shown as SEQ ID NO.4) and NS1-R (shown as SEQ ID NO.5), as well as NS1-T7-F (shown as SEQ ID NO.6) and NS1-T7-R (shown as SEQ ID NO.7).

[0022] In a specific embodiment of the present invention, the kit used for synthesizing the dsRNA is T7 RiboMAX Express RNAi System Kit.

[0023] The steps for obtaining the dsRNA by using in vitro transcription technology are as follows:

[0024] S1. RNA extraction: Extracting the RNA of IPV virus;

[0025] S2. Reverse transcription: Using the obtained RNA as a template and reverse transcribing it into cDNA;

[0026] S3. Synthesizing dsRNA; Using the obtained cDNA as a template, first amplifying the DNA templates for transcribing the sense strand ssRNA and the antisense strand ssRNA respectively by ordinary PCR with the primers NS1-T7F and NS1-R and the primers NS1-F and NS1-T7R, and synthesizing the dsRNA according to the operation instructions of the T7 RiboMAX Express RNAi System Kit.

[0027] The present invention also provides a nucleic acid molecule encoding the dsRNA.

[0028] The present invention also provides an expression cassette containing the nucleic acid molecule.

[0029] The present invention also provides a vector containing the nucleic acid molecule or the expression cassette.

[0030] The present invention also provides a cell capable of expressing the dsRNA, wherein the cell contains the nucleic acid molecule, or contains the expression cassette, or contains the vector.

[0031] The present invention also provides a strain capable of expressing the dsRNA, wherein the strain contains the nucleic acid molecule, or contains the expression cassette, or contains the vector.

[0032] The present invention also provides a reagent containing the dsRNA, the nucleic acid molecule, the expression cassette, the vector, the cell and / or the strain.

[0033] Since the dsRNA of the present invention can significantly inhibit the replication of IPV virus and treat diseases caused by IPV virus infection. Therefore, the present invention claims the following applications of the dsRNA and / or the biological material capable of expressing the dsRNA.

[0034] The present invention claims the application of the dsRNA, the nucleic acid molecule, the expression cassette, the vector, the cell, the strain or the reagent in the preparation of a product for inhibiting infectious precocious puberty virus.

[0035] Specifically, the inhibition is to inhibit the replication of infectious precocious puberty virus.

[0036] The present invention also claims the application of the dsRNA, the nucleic acid molecule, the expression cassette, the vector, the cell, the strain or the reagent in the preparation of a product for preventing and treating viral diseases.

[0037] Specifically, the viral disease is a disease caused by infectious precocious puberty virus infection.

[0038] Optionally, the product in the above application is a drug or a feed additive. For example, the dsRNA can be expressed in bacteria and the bacterial mass can be collected as a feed additive.

[0039] Optionally, the dosage form of the drug is an injection.

[0040] The present invention has the following beneficial effects:

[0041] The present invention provides a dsRNA targeting the NS1 gene of infectious precocious virus. The dsRNA is composed of a sense strand with a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.2. By injecting the dsRNA into Macrobrachium rosenbergii infected with IPV virus, it is found that the use of the dsRNA can significantly reduce the viral load of IPV virus and relieve the growth inhibition of IPV virus on Macrobrachium rosenbergii. Based on this, the dsRNA or biological materials capable of expressing the dsRNA, such as cells or strains capable of expressing the dsRNA, can be used to prepare reagents for inhibiting IPV virus, so as to prevent and treat viral diseases caused by IPV virus infection. The present invention is beneficial to the prevention and treatment of IPV virus-related diseases, reduces the losses of farmers, and promotes the long-term and stable development of the Macrobrachium rosenbergii aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the agarose gel electrophoresis pattern of dsRNA-NS1; Lane 1: DS2000 Marker; Lane 2: dsRNA-NS1.

[0043] Figure 2 It is to detect the expression of the virus in Macrobrachium rosenbergii infected with IPV virus at different time periods after injecting dsRNA-NS1 by using ordinary PCR reaction.

[0044] Figure 3 It is to detect the virus copy number in Macrobrachium rosenbergii infected with IPV virus at different time periods after injecting dsRNA-NS1 by using Taqman probe-based quantitative PCR; **P<0.01 in the figure.

[0045] Figure 4 It is the full-length analysis result of Macrobrachium rosenbergii infected with IPV virus at different time periods after injecting dsRNA-NS1; ***P<0.001; ****P<0.0001.

[0046] Figure 5 It is the body weight analysis result of Macrobrachium rosenbergii infected with IPV virus at different time periods after injecting dsRNA-NS1; ***P<0.001; ****P<0.0001.

[0047] Figure 6 It is the physical sign diagram of Macrobrachium rosenbergii infected with IPV virus on the 56th day after injecting dsRNA-NS1; the upper figure is the top view and the lower figure is the side view.

[0048] Figure 7 It is to detect the expression of the virus in healthy Macrobrachium rosenbergii at different time periods after injecting the crude extract of IPV by using ordinary PCR reaction; the Mock group is the control.

[0049] Figure 8 To detect the viral copy number of healthy Macrobrachium rosenbergii at different time periods after injection of crude IPV extract by quantitative PCR using Taqman probe method; **P<0.01 in the figure**.

[0050] Figure 9 For the full-length analysis of healthy Macrobrachium rosenbergii at different time periods after injection of crude IPV extract; **P<0.01; ***P<0.001; ****P<0.0001**.

[0051] Figure 10 For the body weight analysis of healthy Macrobrachium rosenbergii at different time periods after injection of crude IPV extract; ****P<0.0001**.

[0052] Figure 11 For the physical sign diagram of healthy Macrobrachium rosenbergii on the 56th day after injection of crude IPV extract; the upper figure is the top view and the lower figure is the side view. Specific implementation mode

[0053] The present invention will be further described below in conjunction with the specification drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0055] Example 1 Synthesis of dsRNA targeting the NS1 gene of IPV virus

[0056] Based on the genome of IPV virus, the present invention designed multiple pairs of different dsRNA synthesis primers and synthesized multiple different dsRNAs by in vitro transcription method. Among them, the dsRNA composed only of the sense strand with the nucleotide sequence shown in SEQ ID NO.1 and the antisense strand with the nucleotide sequence shown in SEQ ID NO.2 can effectively inhibit the replication of IPV virus, and the gene it targets is the NS1 gene of IPV virus (the gene DNA sequence is shown in SEQ ID NO.3). Therefore, the present invention names the dsRNA as dsRNA-NS1, and its in vitro transcription synthesis process is as follows:

[0057] 1. Primer design

[0058] The primer pairs designed for synthesizing dsRNA-NS1 in the present invention include the specific primer NS1-F (shown in SEQ ID NO.4) and NS1-R (shown in SEQ ID NO.5), as well as the specific primers NS1-T7-F (shown in SEQ ID NO.6) and NS1-T7-R (shown in SEQ ID NO.7) containing the T7 sequence. In addition, the present invention also designs specific primers for synthesizing dsRNA targeting the gene encoding green fluorescent protein (GFP).

[0059] The nucleotide sequences of the above-mentioned primers are shown in Table 1.

[0060] Table 1 Specific primers for synthesizing dsRNA

[0061]

[0062]

[0063] 2. In vitro transcription of dsRNA

[0064] (1) RNA extraction: Using the Eastep Super total RNA extraction kit (Promega), extract the RNA of the gill tissue of Macrobrachium rosenbergii detected to be infected with IPV virus according to its instruction manual; measure the concentration of the extracted RNA, and at the same time detect the quality of the extracted RNA with 1% agarose gel, and store it at -80°C;

[0065] (2) Reverse transcription: Using the obtained RNA as a template, reverse transcribe it into cDNA according to the instruction manual of the PrimeScript RT reagent Kit with gDNAEraser (Perfect Real Time) kit;

[0066] (3) Synthesize dsRNA-NS1; Using the obtained cDNA as a template, first amplify the DNA templates for transcribing the sense strand ssRNA and the antisense strand ssRNA by ordinary PCR with the primers NS1-T7F and NS1-R and the primers NS1-F and NS1-T7R respectively, and synthesize dsRNA-NS1 according to the instruction manual of the T7 RiboMAX Express RNAi System Kit; dsRNA-GFP (dsGFP) is synthesized using the GFP plasmid as a template; measure the concentration of the synthesized dsRNA, detect its integrity by electrophoresis, and store it at -80°C.

[0067] A more specific method for synthesizing dsRNA-NS1 is as follows:

[0068] a. Obtaining the DNA template for transcription by ordinary PCR: Using the cDNA of the obtained IPV virus as a template, through ordinary PCR, with NS1-T7F and NS1-R primers, and NS1-F and NS1-T7R primers respectively, amplify the DNA templates for transcribing the sense strand ssRNA and the antisense strand ssRNA, and perform gel recovery and purification;

[0069] b. Transcribing ssRNA: Respectively use the DNA templates for transcribing the sense strand ssRNA and the antisense strand ssRNA obtained in a to transcribe the sense strand ssRNA and the antisense strand ssRNA; The reaction system is shown in the following table, and the reaction conditions are 37 °C for 4 hours;

[0070]

[0071] c. Annealing of dsRNA and removal of ssRNA: Mix equal volumes of the complementary RNA reaction solutions, incubate at 70 °C for 10 minutes, and slowly cool to room temperature to achieve annealing of double-stranded RNA; Add 1 μL of RNase to 199 μL of nuclease-free water to dilute the accompanying RNase solution (1:200); For every 20 μL of reaction volume, add 1 μL of freshly diluted RNase solution and 1 μL of RQ1 RNase-Free DNase, and incubate at 37 °C for 30 minutes to remove all residual single-stranded RNA and DNA templates, leaving only double-stranded RNA;

[0072] d. Purifying dsRNA: Add 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of 95% ethanol, mix well and place on ice for 5 minutes; Centrifuge at 16000g for 10 minutes, remove the supernatant, air-dry the precipitate and resuspend it in 100 μL of nuclease-free water to complete the purification of dsRNA; Take 1 μL of dsRNA, dilute it 20 times with nuclease-free water, detect the concentration and perform agarose gel detection.

[0073] The agarose gel electrophoresis pattern of dsRNA-NS1 is as Figure 1 shown; Lane 1: DS2000 Marker; Lane 2: dsRNA-NS1. It can be Figure 1 seen that the present invention obtains dsRNA-NS1 by in vitro transcription.

[0074] Example 2 Effect of dsRNA-NS1 treatment on IPV virus replication

[0075] 1. Preparation of crude IPV virus extract

[0076] The crude IPV virus extract is derived from the gill tissues of Macrobrachium rosenbergii that have been detected to carry the IPV virus, and the crude extract used for the control group is derived from the gill tissues of healthy Macrobrachium rosenbergii that have been detected not to carry the IPV virus.

[0077] Take a number of fresh gill tissues and place them in a 5 mL special grinding tube containing 1×PBS solution; add 2 special grinding steel balls to each grinding tube, set the parameters of the grinder to -30°C and 50 HZ, and the grinding time is 10 min. Then seal the grinding tube and put it into the grinder to fully shake and grind into a homogenate; centrifuge the homogenate, set the conditions to 8000 g and 4°C, and the centrifugation time is 20 min. Take the supernatant and filter it through a 0.22 μm filter membrane to obtain the crude virus extract and the crude extract of healthy shrimp.

[0078] 2. Infection of Macrobrachium rosenbergii: Use a sterile insulin syringe (1 mL) to aspirate 75 μL of the crude IPV virus extract (the IPV virus copy number is 6.84×10 7 copies / tail) and inject it into the second abdominal muscle of Macrobrachium rosenbergii, and the infection duration is 14 days.

[0079] 3. dsRNA treatment: Divide the obtained Macrobrachium rosenbergii infected with the IPV virus into two groups, namely the experimental group (IPV+dsNS1 group) and the control group (IPV+dsGFP group), with 20 tails in each group, ensuring that the vitality status of the two groups of shrimp is the same and there is no significant difference in body weight and total length. Before injection, dilute the dsRNA with sterilized 1×PBS, and inject 50 μL of dsNS1 (the dsRNA injection amount is 4 μg / g of shrimp) into the shrimp in the IPV+dsNS1 group on the 0th, 2nd, 7th, 14th, 28th, and 42nd days, and inject an equal amount of dsGFP into the shrimp in the IPV+dsGFP group; use a sterile insulin syringe (1 mL) to inject the second abdominal muscle of the shrimp.

[0080] 4. Virus detection: On the 0th, 7th, 14th, 28th, 42nd, and 56th days after the first injection of dsRNA, take the base of the swimming legs of Macrobrachium rosenbergii (one shrimp is one sample, a total of 3 samples). Select the Eastep Super total RNA extraction kit to extract tissue RNA and operate according to the instructions. After extracting the RNA, measure its concentration and reverse transcribe it into cDNA according to the instructions of the PrimeScript RT reagent Kit with gDNAEraser(Perfect Real Time) kit.

[0081] Using the obtained cDNA as a template, perform a PCR reaction to detect the expression of IPV virus using ordinary PCR, and simultaneously detect the virus copy number by quantitative PCR with Taqman probe method. The primers used in the ordinary PCR reaction are IPV-F and IPV-R (shown in Table 2), the ordinary PCR reaction system is shown in Table 3, and the ordinary PCR reaction program is shown in Table 4.

[0082] Table 2 Primer Sequences

[0083]

[0084] Table 3 Ordinary PCR Reaction System

[0085]

[0086] Table 4 Ordinary PCR Reaction Program

[0087]

[0088]

[0089] After the reaction, perform electrophoresis detection.

[0090] Using the ordinary PCR reaction to detect the expression of the virus in Macrobrachium rosenbergii infected with IPV virus at different time periods after injection of dsRNA-NS1 is as Figure 2 shown. As can be Figure 2 seen, on the 0th day, brighter target bands were amplified in both groups; in the IPV+dsGFP group, target bands were detected in all 5 detections from 7 to 56 days; while in the IPV+dsNS1 group, the target band amplified from the sample on the 7th day was faint, and no target bands were amplified in the dsNS1 group in the 4 detections from 14 to 56 days, indicating that dsRNA-NS1 can successfully knockdown the IPV virus.

[0091] Further detect the IPV virus concentration by quantitative PCR with Taqman probe method. The primers used in the quantitative PCR with Taqman probe method are IPV-T-qF and IPV-T-qR (as shown in Table 2), and the probe used is IPV-T-probe (as shown in Table 2).

[0092] Operate according to the instructions of the Probe qPCR Mix kit (purchased from Takara). The data is expressed in the form of mean ± standard deviation, and statistical significance is analyzed using Student's t-test.

[0093] Using the quantitative PCR with Taqman probe method to detect the virus copy number in Macrobrachium rosenbergii infected with IPV virus at different time periods after injection of dsRNA-NS1 is as Figure 3 shown. As can be Figure 3It can be seen that the virus copy numbers of the IPV+dsGFP group and the IPV+dsNS1 group were 10 6.75±0.20 copies / μg and 10 6.64±0.17 copies / μg on day 0 and day 7 respectively, and 10 6.38±0.45 copies / μg and 10 5.64 ±0.23 copies / μg on day 14 respectively. There was no significant difference between the two groups. The virus copy numbers of the IPV+dsGFP group and the IPV+dsNS1 group were 10 6.28±0.83 copies / μg and 10 3.89±0.15 copies / μg on day 14 respectively, showing a significant difference. The virus copy numbers of the IPV+dsGFP group were 10 6.51±0.19 copies / μg, 10 6.94±0.44 copies / μg, and 10 7.52± 0.23 copies / μg on day 28, day 42, and day 56 respectively, while the virus copy number of the IPV+dsNS1 group was not detected from day 28 to day 56, indicating that the effect of dsRNA-NS1 in knocking down IPV virus was significant.

[0094] Example 3 Effect of dsRNA-NS1 treatment on the growth of Macrobrachium rosenbergii infected with IPV virus

[0095] When conducting the experiment described in Example 2, in addition to sampling and detecting the virus copy number of IPV, the present invention measured the total length and body weight of Macrobrachium rosenbergii on day 0, day 14, day 28, day 42, and day 56 after the first injection of dsRNA.

[0096] The analysis results of the total length of Macrobrachium rosenbergii infected with IPV virus at different time periods after injection of dsRNA-NS1 are as Figure 4 shown. It can be seen from Figure 4 that on day 0, the initial total lengths of Macrobrachium rosenbergii in the IPV+dsGFP group and the IPV+dsNS1 group were 5.22±0.16 cm and 5.19±0.15 cm respectively. On day 14, the total lengths of the two groups were 5.57±0.21 cm and 5.60±0.21 cm respectively, and no difference was shown yet. However, on day 28, the total lengths of the two groups were 6.00±0.28 cm and 6.20±0.37 cm respectively. On day 42, the total lengths of the two groups were 6.35±0.35 cm and 6.67±0.42 cm respectively. On day 56, the total lengths of the two groups were 6.75±0.36 cm and 7.08±0.42 cm respectively, all showing significant differences.

[0097] The analysis results of the body weight of Macrobrachium rosenbergii infected with IPV virus at different time periods after injection of dsRNA-NS1 are as Figure 5As shown. By Figure 5 It can be seen that on the 0th day, the initial weights of Macrobrachium rosenbergii in the IPV+dsGFP group and the IPV+dsNS1 group were 1.44±0.08 g and 1.44±0.08 g respectively, with no significant difference; on the 14th day, the weights of the two groups were 1.7±0.20 g and 1.71±0.122 g respectively, and no difference was shown yet; but on the 28th day, the weights of the two groups were 2.12±0.26 g and 2.28±0.42 g respectively, on the 42nd day the weights of the two groups were 2.56±0.41 g and 2.94±0.53 g respectively, and on the 56th day the weights of the two groups were 3.04±0.43 and 3.44±0.71 g respectively, all with significant differences.

[0098] The physical signs of Macrobrachium rosenbergii infected with IPV virus on the 56th day after injection of dsRNA-NS1 are as Figure 6 shown. By Figure 6 It can be seen that compared with the IPV+dsGFP group, the individuals in the IPV+dsNS1 group were significantly larger, indicating that the injection of dsRNA-NS1 relieved the growth inhibition of IPV virus on Macrobrachium rosenbergii.

[0099] Example 4 Detection of the virulence effect of IPV virus

[0100] Before detecting the effects of dsRNA-NS1 on IPV virus replication, etc., the present invention detected the virulence effect of the prepared crude IPV virus extract. That is, after injecting the crude IPV virus extract into Macrobrachium rosenbergii (using a sterile insulin syringe (1 mL) to aspirate 75 μL of the crude IPV virus extract (the copy number of IPV virus is 6.84×10 7 copies / tail) and injecting it into the second abdominal muscle of Macrobrachium rosenbergii), in order to monitor the IPV virus infection effect, the infection situation of IPV virus was qualitatively and quantitatively detected by ordinary PCR and Taqman probe-based quantitative PCR respectively. At the same time, the total length and weight of Macrobrachium rosenbergii were measured every 14 days.

[0101] After injecting the crude IPV virus extract, samples were taken once every 7 days in the first 14 days, and once every 14 days after 14 days. The expression of the virus in healthy Macrobrachium rosenbergii at different time periods after injection of the crude IPV extract was detected by ordinary PCR reaction as Figure 7 shown; the Mock group was the control without injection of IPV virus. By Figure 7 It can be seen that on the 0th day after injection of the crude IPV extract, no target band was amplified in both the Mock control group and the IPV infection group; on the 7th day, only a faint target band was amplified in the IPV group, and in the subsequent 4 detections, only a brighter target band was detected in the IPV group. The virus copy number in healthy Macrobrachium rosenbergii at different time periods after injection of the crude IPV extract was detected by Taqman probe-based quantitative PCR as Figure 8As shown in. From Figure 8 it can be seen that in the Mock group, the virus copy number could not be detected in all 6 detections, indicating that the shrimp were healthy and pollution-free; in the IPV group, the virus copy number could not be detected on the 0th day, and on the subsequent 7th day (10 4.55±0.25 copies / μg), 14th day (10 5.94±0.13 copies / μg), 28th day (10 7.68±0.19 copies / μg), 42nd day (10 8.03±0.44 copies / μg), and 56th day (10 8.2±0.27 copies / μg), the virus copy number gradually increased.

[0102] The results of the full-length analysis and weight analysis of healthy Macrobrachium rosenbergii at different time periods after injection with the crude IPV extract are shown in Figure 9 and Figure 10 respectively. From Figure 9 it can be seen that on the 0th day, there were no significant differences in the initial full length (6.26 ± 0.15 cm and 6.27 ± 0.16 cm) and initial weight (2.43 ± 0.12 g and 2.44 ± 0.14 g) of the shrimp in the Mock and IPV groups. The full lengths of the Mock and IPV groups were 7.04 ± 0.33 cm and 6.88 ± 0.22 cm, 7.86 ± 0.32 cm and 7.58 ± 0.47 cm, 8.46 ± 0.39 cm and 8.06 ± 0.45 cm, 8.89 ± 0.56 cm and 8.51 ± 0.45 cm on the 14th day, 28th day, 42nd day, and 56th day respectively, and there were significant differences. From Figure 10 it can be seen that the weights of the shrimp in the Mock and IPV groups were 3.57 ± 0.41 g and 3.33 ± 0.32 g, 5.10 ± 0.76 g and 4.56 ± 0.91 g, 6.55 ± 1.05 g and 5.69 ± 1.02 g, 7.89 ± 1.67 g and 6.69 ± 1.51 g on the 14th day, 28th day, 42nd day, and 56th day respectively, and there were significant differences.

[0103] The physical signs of healthy Macrobrachium rosenbergii on the 56th day after injection with the crude IPV extract are shown in Figure 11 As shown in. From Figure 11 it can be seen that compared with the Mock group, the individuals in the IPV group were smaller, indicating that the growth of Macrobrachium rosenbergii would be inhibited after infection with the IPV virus.

[0104] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A dsRNA targeting the NS1 gene of an infectious precocious virus, characterized in that: The dsRNA consists of a sense strand with a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.

2.

2. A nucleic acid molecule encoding the dsRNA of claim 1.

3. An expression cassette containing the nucleic acid molecule of claim 2.

4. A vector comprising the nucleic acid molecule of claim 2 or the expression cassette of claim 3.

5. A cell capable of expressing the dsRNA according to claim 1, characterized in that: The cell contains the nucleic acid molecule according to claim 2, or the expression cassette according to claim 3, or the vector according to claim 4.

6. A strain capable of expressing the dsRNA according to claim 1, characterized in that: The strain contains the nucleic acid molecule according to claim 2, or the expression cassette according to claim 3, or the vector according to claim 4.

7. A reagent comprising the dsRNA of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, the vector of claim 4, the cell of claim 5 and / or the strain of claim 6.

8. Use of the dsRNA of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, the vector of claim 4, the cell of claim 5, the strain of claim 6 or the reagent of claim 7 in the preparation of a product for inhibiting infectious precocious virus.

9. The use according to claim 8, characterized in that: The inhibition is the inhibition of the replication of infectious premature virus.

10. Use of the dsRNA of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, the vector of claim 4, the cell of claim 5, the strain of claim 6 or the agent of claim 7 in the preparation of a product for preventing and treating viral diseases, characterized in that: The viral disease is a disease caused by infection with an infectious precocious virus.

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