AmiRNA for inhibiting porcine rotavirus and its application

Abstract

The present invention belongs to the technical field of genetic engineering and vaccine development research, and specifically relates to an amiRNA for inhibiting porcine rotavirus and its application. The present invention provides an amiRNA for inhibiting porcine rotavirus, including amiRNA-323 or amiRNA-345. The amiRNA provided by the present invention can effectively inhibit porcine rotavirus, especially the PORV-GD-01-2015 strain. It is verified by the examples that the amiRNA provided by the present invention can effectively inhibit the replication and proliferation of the porcine rotavirus PORV-GD-01-2015 strain, and can also be used in combination with antiviral drugs and used for the cultivation of porcine rotavirus disease resistance system, which provides an effective method for the development of amiRNA vaccines to treat porcine rotavirus, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and vaccine development research, and specifically relates to an amiRNA for inhibiting porcine rotavirus and an application thereof. Background Art

[0002] Porcine rotavirus (PORV) belongs to the family Reoviridae and is characterized by double-stranded RNA (dsRNA) containing 11 genomic segments and a capsid composed of three concentric protein layers. Porcine group A rotavirus (PoRVA) is the leading cause of neonatal diarrhea in suckling piglets and newly weaned piglets worldwide. Rotaviruses often co-infect with other viral agents, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and bacterial pathogens, such as Escherichia coli, causing significant losses to the pig industry. In addition, the evolution of human rotaviruses is closely intertwined with animal rotaviruses, and porcine and bovine rotaviruses are considered important animal pathogens because of their widespread economic impact in livestock and their potential sources of heterologous rotavirus infections in humans. Therefore, the control and prevention of porcine rotavirus is a key step in rotavirus purification and ecological safety.

[0003] The rotavirus genome consists of 11 double-stranded RNA segments ranging in size from 0.6kb to 3.3kb, encoding 12 proteins. Vp4 and Vp7 constitute the outer shell components, Vp6 is an intermediate layer protein, and the structural proteins Vp1, Vp2, and Vp3 constitute the core. The remaining proteins, Nsp1, Nsp2, Nsp3, NSP4, Nsp5, and Nsp6 are non-structural proteins. Vp4 is a non-glycosylated trypsin-sensitive protein with receptor binding activity, Vp6 is a group- and subgroup-specific antigen, and the epitopes contained on the surface of Vp7 are recognized by cytotoxic T cells. According to the antigenicity of Vp4 and Vp7, group A rotavirus can be divided into P and G genotypes. Group A porcine rotavirus includes 7 P genotypes and 10 G genotypes. Due to the diversity of rotavirus genotypes, it exhibits strong survival ability in the environment and is insensitive to many chemical disinfectants and preservatives. Currently, there is no vaccine on the market that can effectively inhibit rotavirus. Farmers can only frequently use antibiotics and fluid therapy to reduce the clinical symptoms of secondary infection after rotavirus infection. Frequent use of antibiotics makes the bacteria more and more resistant, and the efficacy of antibiotics is gradually reduced or even ineffective. Therefore, providing an amiRNA that can effectively inhibit the replication of the dominant genotype of porcine rotavirus to prevent and control diseases directly or indirectly caused by porcine rotavirus and exploring technologies for prevention and treatment of different strains has great scientific significance and application prospects. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an amiRNA that can effectively inhibit porcine rotavirus, so as to achieve the prevention and treatment of diseases caused by porcine rotavirus.

[0005] The present invention provides an amiRNA for inhibiting porcine rotavirus, wherein the amiRNA comprises amiRNA-323 or amiRNA-345;

[0006] The nucleotide sequence of the target of amiRNA-323 is shown in SEQ ID No.5;

[0007] The nucleotide sequence of the target of amiRNA-345 is shown in SEQ ID No.8.

[0008] Preferably, the nucleotide sequence of the sense strand of the amiRNA-323 is shown as SEQ ID No. 1, and the nucleotide sequence of the antisense strand is shown as SEQ ID No. 2;

[0009] The nucleotide sequence of the sense strand of the amiRNA-345 is shown in SEQ ID No.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID No.4.

[0010] The present invention also provides the use of the amiRNA described in the above technical solution in the preparation of products for preventing and treating diseases caused by porcine rotavirus.

[0011] Preferably, the porcine rotavirus comprises the PORV-GD-01-2015 strain.

[0012] Preferably, the product comprises a biological product.

[0013] Preferably, the biological product comprises a vaccine.

[0014] The present invention also provides a vaccine for preventing and / or treating diseases caused by porcine rotavirus, wherein the active ingredient of the vaccine comprises the amiRNA described in the above technical solution.

[0015] The present invention also provides the use of the amiRNA described in the above technical solution in preparing a transgenic cell line resistant to porcine rotavirus.

[0016] The present invention also provides the use of the amiRNA described in the above technical solution in breeding transgenic pigs resistant to porcine rotavirus.

[0017] The present invention also provides the application of the amiRNA described in the above technical solution in the study of the infection mechanism of porcine rotavirus by VP7 and VP6 genes.

[0018] Beneficial Effects

[0019] The amiRNA provided by the present invention can effectively inhibit porcine rotavirus, especially the PORV-GD-01-2015 strain. It has been verified by the examples that the amiRNA-323 and amiRNA-345 provided by the present invention can effectively inhibit the replication and proliferation of the porcine rotavirus PORV-GD-01-2015 strain, and can also be used in combination with antiviral drugs and used for the cultivation of porcine rotavirus disease resistance system, which provides an effective method for the development of amiRNA vaccine to treat porcine rotavirus and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 Schematic diagram of the structure of amiRNA expression vector;

[0022] Figure 2 The cytopathic effect of normal Vero cells not transfected with amiRNA 48 hours after infection (100× microscopic examination result);

[0023] Figure 3 The cytopathic effect of Vero cells transfected with empty plasmid 48 hours after infection (100× microscopic examination result);

[0024] Figure 4 The cytopathic effect of Vero cells transfected with pcDNA6.2-VP7-323 48 hours after infection (100× microscopic examination results);

[0025] Figure 5 The cytopathic effect of Vero cells transfected with pcDNA6.2-VP7-345 48 hours after infection (100× microscopic examination results);

[0026] Figure 6 This is an electron microscopic picture of normal cells that have not been transfected or infected (100× microscopic examination result);

[0027] Figure 7 The results of PORV virus titer detection in amiRNA-expressing cells infected with PORV-GD-01-2015 strain;

[0028] Figure 8 Indirect immunofluorescence of IFA after transfection with plasmid pcDNA6.2-VP7-345;

[0029] Fig. 9 Indirect immunofluorescence of IFA after transfection with plasmid pcDNA6.2-VP7-323;

[0030] Fig.10 The indirect immunofluorescence results of IFA in cells not transfected with artificial amiRNA after infection;

[0031] Fig.11 The indirect immunofluorescence of IFA after transfection with empty plasmid;

[0032] Fig.12 This is the indirect immunofluorescence of IFA of normal untransfected and unvaccinated cells. DETAILED DESCRIPTION

[0033] The present invention provides an amiRNA for inhibiting porcine rotavirus, wherein the amiRNA includes amiRNA-323 or amiRNA-345; the nucleotide sequence of the target of the amiRNA-323 is shown in SEQ ID No. 5, specifically: 5'-GTAAGACGGATGGGTATCTAA-3';

[0034] The nucleotide sequence of the target of amiRNA-345 is shown in SEQ ID No. 8, specifically: 5'-CAGAAATGGAATGCAACCTCA-3'.

[0035] In the present invention, the nucleotide sequence of the sense strand of the amiRNA-323 is preferably as shown in SEQ ID No. 1, specifically: 5'-TGCTGTTAGATACCCATCCGTCTTACGTTTTGGCCACTG ACTGACGTAAGACGTGGGTATCTAA-3'; the nucleotide sequence of the antisense strand of the amiRNA-323 is preferably as shown in SEQ ID No. 2, specifically: 5'-CCTGTTAGATACCCACGTCTTAC GTCAGTCAGTGGCCAAAACGTAAGACGGATGGGTATCTAAC-3'.

[0036] In the present invention, the nucleotide sequence of the sense strand of the amiRNA-345 is preferably as shown in SEQ ID No.3, specifically: 5'-TGCTGTGAGGTTGCATTCCATTTCTGGTTTTGGCCACTG ACTGACCAGAAATGATGCAACCTCA-3'; the nucleotide sequence of the antisense strand of the amiRNA-345 is preferably as shown in SEQ ID No.4, specifically: 5'-CCTGTGAGGTTGCATCATTTC TGGTCAGTCAGTGGCCAAAACCAGAAATGGAATGCAACCTCAC-3'.

[0037] The present invention constructs amiRNA targeting the conservative sequences of PORV VP7 and VP6 genes. Compared with siRNA targeting PORV protein coding genes, amiRNA can inhibit the replication and proliferation of PORV, and can be used not only for the development of new biological vaccine preparations against PORV, but also for the breeding of disease-resistant pigs in the later stage.

[0038] The present invention also provides the use of the amiRNA described in the above technical solution in the preparation of products for preventing and treating diseases caused by porcine rotavirus.

[0039] In the present invention, the porcine rotavirus preferably includes the PORV-GD-01-2015 strain (for strain information, refer to: Yang Juan. Analysis of the complete gene sequence of porcine rotavirus GD-01-2015 and establishment of a double sandwich ELISA method [D]. Yangzhou University, 2018.). The product of the present invention preferably includes a biological product; the biological product preferably includes a vaccine.

[0040] The present invention also provides a vaccine for preventing and / or treating diseases caused by porcine rotavirus, wherein the active ingredient of the vaccine comprises the amiRNA described in the above technical solution.

[0041] The present invention also provides the use of the amiRNA described in the above technical solution in preparing a transgenic cell line resistant to porcine rotavirus.

[0042] The present invention also provides the use of the amiRNA described in the above technical solution in breeding transgenic pigs resistant to porcine rotavirus.

[0043] The present invention also provides the application of the amiRNA described in the above technical solution in the study of the infection mechanism of porcine rotavirus by VP7 and VP6 genes.

[0044] To further illustrate the present invention, an amiRNA for inhibiting porcine rotavirus and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] In the embodiment of the present invention, the pcDNA6.2-GW / EmGFP-miR vector was purchased from Invitrogen Life Technologies, Inc., USA; the PORV-GD-01-2015 strain was isolated and preserved by this laboratory (for strain information, refer to: Yang Juan. Full gene sequence analysis of porcine rotavirus GD-01-2015 and establishment of double sandwich ELISA method [D]. Yangzhou University, 2018.).

[0046] Example 1

[0047] miRNA RNAi design

[0048] Using bioinformatics methods, the VP7 and VP6 gene sequences of PORV on NCBI were compared for homology, and some of their conserved sequences were selected as the amiRNA target regions of PORV. The conserved sequence fragments are shown in Table 1, where the starting position refers to the corresponding base site on the nucleotide sequence with accession number AB905221.1.

[0049] Table 1 PORV VP7 / VP6 gene target sequences

[0050]

[0051] According to Invitrogen's BLOCK-iT TM The instruction manual of Inducible Pol II miR RNAi Expression Vector Kit with EmGFP provides the design principles of artificial miRNA. Artificial miRNA sequences targeting PORV are designed with the four groups of gene sequences in Table 1 as targets, and double-stranded oligonucleotide sequences of artificial miRNA expression plasmids are constructed, as shown in Table 2.

[0052] Table 2 Double-stranded oligonucleotide sequences for artificial miRNA expression

[0053]

[0054] The sequences shown in Table 2 were sent to Beijing Qingke Company for single-stranded oligonucleotide (Oligo) synthesis. Each pair of single-stranded oligonucleotides was annealed, and the double-stranded oligonucleotide sequence of the artificial miRNA expression plasmid was constructed; the reaction system was: 5 μL of positive-strand DNA Oligo (200 μM), 5 μL of negative-strand DNA Oligo (200 μM), 2 μL of 10×Oligo Annealing Buffer (100 mMTris-HCl PH 8.0, 500 mM NaCl, 10 mM EDTA), and 8 μL of deionized water. The reaction system was incubated at 94°C for 5 minutes, and then slowly cooled to room temperature for annealing, and the artificial miRNA expression plasmid double-stranded oligonucleotide sequences VP6-207F / R, VP6-345F / R, VP7-323F / R and VP7-487F / R were obtained.

[0055] The target sequence is screened through preliminary experiments. The specific steps are as follows:

[0056] (1) Dilution of single-stranded dry powdered oligo

[0057] The dry powdered Oligo was centrifuged at 12000 rpm for 1 min, vortexed, and centrifuged at 12000 rpm for 1 min to separate into four groups: VP6-207, VP6-345, VP7-323, and VP7-487.

[0058] In the VP6-207 group, 8.35 μL VP6-207F / R was added to make the final concentration 200 μM and mixed;

[0059] In the VP6-345 group, 8.35 μL VP6-345F / R was added to make the final concentration 200 μM and mixed;

[0060] In the VP7-323 group, 8.35 μL VP7-323F / R was added to make the final concentration 200 μM and mixed;

[0061] In the VP7-487 group, 8.35 μL VP7-487F / R was added to make the final concentration 200 μM and mixed.

[0062] (2) Generation of double-stranded oligo (concentration 50 μM)

[0063] The reaction system was prepared for each group as follows:

[0064] VP6-207 group: 5 μL VP6-207F / R (200 μM), 2 μL 10×OligoAnnealing buffer, 8 μL DNase / RNase free water, total system 20 μL;

[0065] VP6-345 group: 5 μL VP6-345F / R (200 μM), 2 μL 10×OligoAnnealing buffer, 8 μL DNase / RNase free water, total system 20 μL;

[0066] VP7-323 group: 5 μL VP7-323F / R (200 μM), 2 μL 10×OligoAnnealing buffer, 8 μL DNase / RNase free water, total system 20 μL;

[0067] VP7-487 group: 5 μL VP7-487F / R (200 μM), 2 μL 10×OligoAnnealing buffer, 8 μL DNase / RNase free water, total system 20 μL;

[0068] After the reaction system is prepared, gently blow to mix, centrifuge briefly, and incubate at 95°C in a PCR instrument for 4 minutes, then cool at room temperature for 5 to 10 minutes.

[0069] (3) 50μM double-stranded oligo was diluted to 500nM

[0070] Add 1 μL of 50 μM double-stranded oligo to 99 μL of DNase / RNase free water to obtain 500 nM double-stranded oligo.

[0071] (4) 500nM double-stranded oligo was diluted to 10nM

[0072] Add 44 μL of DNase / RNase free water, 1 μL of 500 nM double-stranded oligo, and 5 μL of 10×Oligo Annealing buffer to each group of PCR tubes in sequence, with a total volume of 50 μL.

[0073] (5) Connecting carrier

[0074] Add DNase / RNase free water 4μL, 5× Ligation Buffer 2μL, pcDNA TM 6.2-GW / EmGFP-miR, linearized 1μL, 10nM double-stranded oligo 2μL, T4 DNA Ligase 1μL, total volume 10μL. Gently pipette to mix, place at 4℃ overnight to obtain the ligation product.

[0075] (6) Transformation of Ligation Product

[0076] Take 5 μL of the ligation product from each group and add it to 50 μL DH5α, flick to mix, and let it stand on ice for 30 min; heat shock at 42°C for 30 min; let it stand on ice for 3 min; add 700 μL of LB liquid culture medium without resistance to the tube; place it in a 37°C shaker at 200 rpm for 1 h; centrifuge it at 5000 rpm for 3 min at room temperature; discard the supernatant, add 200 μL of LB liquid containing ampicillin resistance, resuspend the plate, and place it in a 37°C incubator for 12 to 14 h.

[0077] (7) Colony expansion and identification

[0078] The next day, pick a single colony into a test tube containing 5 mL of ampicillin-resistant LB liquid and culture it in a 37°C shaker at 200 rpm for 14-16 hours. Then extract the plasmid. If the plasmid concentration is too low, it means that the transformed connection vector cannot be stably maintained as the bacteria replicate. Therefore, select a plasmid with a concentration greater than 100 ng / μL for sequencing identification and subsequent transfection experiments.

[0079] The final plasmid concentrations obtained by transformation of amiRNA-323 (SEQ ID No.5) and amiRNA-345 (SEQ ID No.8) were both greater than 100 ng / μL, indicating that amiRNA-323 (SEQ ID No.5) and amiRNA-345 (SEQ ID No.8) can be successfully constructed to ensure that amiRNA plasmids can be stably transfected into cells and have an inhibitory effect on viral replication. Therefore, amiRNA-323 (SEQ ID No.5) and amiRNA-345 (SEQ ID No.8) were finally selected as target sequences.

[0080] The double-stranded oligonucleotides for expression of amiRNA-323 and amiRNA-345 in Table 2 were cloned into pcDNA TM 6.2-GW / EmGFP-miR vector, the insertion site is as follows Figure 1 As shown, the ligation reaction product was transformed into DH5α competent cells.

[0081] Figure 1 The sequence of 5'→3' is as follows: The first 5 nucleotide sequences of the sense strand (TGCTG) are derived from endogenous microRNA-155 and belong to the miRNA class of molecules. The 5' end of the antisense strand contains a 4-nucleotide overhang sequence that corresponds to the 4-nucleotide overhang on the linear structure of the pcDNA6.2-GW / EmGFP-miR plasmid vector, and these sequences can form a double-stranded structure. The 21 nucleotide sequence following the sense strand is a mature miRNA sequence. After transcription, it will complementarily pair with the target RNA, so it is necessary to ensure that it is complementary to the target RNA during design.

[0082] Then the positive plasmids were screened, monoclonal colonies were selected for amplification and culture, and plasmids were extracted. The extracted positive plasmids were first identified by enzyme digestion, and the plasmids with correct enzyme digestion identification (bands of 5608bp and 151bp) were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing using EmGFP forward sequencing primer and miRNA reverse sequencing primer. The plasmids with correct sequencing were retained, and the plasmid containing the nucleotide sequence of amiRNA-323 was named pcDNA6.2-VP7-323, and the plasmid containing the nucleotide sequence of amiRNA-345 was named pcDNA6.2-VP6-345.

[0083] Test Example 1

[0084] Transfection

[0085] One day before transfection, Vero cells were seeded on cell plates at a density of 2 × 10 5 The Vero cells were cultured in DMEM containing 10% fetal bovine serum until the cell density reached 70% to 80%. The Vero cells were then divided into a control group, an amiRNA-323 group, and an amiRNA-345 group.

[0086] Among them, the control group: the 6-well cell plate was placed in a cell culture incubator at 37°C and 5% CO2 for incubation for 6 hours, and then replaced with a maintenance solution containing 2% fetal bovine serum; the timing was started from the beginning of incubation, and after 24 to 48 hours, each well was inoculated with PORV at an MOI of 0.01, and 2 hours after virus infection, a maintenance solution with a final concentration of 5 μg / mL pancreatin was added;

[0087] AmiRNA-323 group: According to the instructions of LipofectamineTM3000, the constructed plasmid pcDNA6.2-VP7-323 was transfected, and the 6-well cell plate was placed in a cell culture incubator at 37°C and 5% CO2 for 6 hours, then the cell status was observed and the culture medium was replaced. If the cell status was not good, it was replaced with DMEM culture medium containing 5% fetal bovine serum; if the cell status was good, it was replaced with DMEM culture medium containing 2% fetal bovine serum. 24 to 48 hours after plasmid transfection, PORV with MOI = 0.01 was inoculated in each well, and 2 hours after virus infection, a maintenance solution with a final concentration of 5μg / mL trypsin was added;

[0088] AmiRNA-345 group: According to the instructions of LipofectamineTM3000, the constructed plasmid pcDNA6.2-VP6-345 was transfected, and the 6-well cell plate was placed in a cell culture incubator at 37°C and 5% CO2 for 6 hours, and then the cell state was observed and the culture medium was replaced. If the cell state was not good, it was replaced with DMEM culture medium containing 5% fetal bovine serum; if the cell state was good, it was replaced with DMEM culture medium containing 2% fetal bovine serum. 24-48 hours after plasmid transfection, PORV with MOI = 0.01 was inoculated in each well, and maintenance solution containing 5μg / mL trypsin was added 2 hours after virus infection.

[0089] PORV replication inhibition effect detection:

[0090] 1. The constructed plasmids amiRNA-VP7-323 and amiRNA-VP6-345 were fluorescently labeled. The fluorescence was observed under a fluorescence microscope to determine the transfection efficiency. When the transfection efficiency was the highest, the PORV-GD-01-2015 strain was used to infect Vero cells at an infection index MOI of 0.01. 48 hours after infection, the cytopathic effect was observed under an electron microscope. The results are as follows: Figure 2 to Figure 6 shown.

[0091] Depend on Figure 2 to Figure 6 It can be seen that Vero cells transfected with amiRNA-VP7-323 and amiRNA-VP6-345 showed less cytopathic effect. The degree of cytopathic effect in the group transfected with amiRNA empty plasmid was consistent with that of positive cells, and the general pathological manifestations were cell rounding, increased refractive index, the appearance of a net-pulling phenomenon, cell rupture, and typical CPE.

[0092] 2. Scrape the cells 48 hours after PORV infection, centrifuge at 12000rpm for 10min, collect the supernatant, and inoculate on Vero cells for virus TCID 50 The results were as follows Figure 7 And as shown in Table 3. Figure 7 The vertical axis represents the virus titer (TCID 50 ); pcDNA6.2-VP7-323 represents cells transfected with an artificial miRNA expression vector with PORV VP7 gene 323 site as the starting site; pcDNA6.2-VP7-345 represents cells transfected with an amiRNA expression vector with PORV VP6 gene 345 site as the starting site; pcDNA6.2-negative represents an empty-load transfected cell control; virus control represents a non-transfected cell virus-infected control; and cell control represents a non-transfected cell virus-infected control.

[0093] Table 3 PORV virus titer detection results in amiRNA-expressing cells

[0094]

[0095] Depend on Figure 7 As shown in Table 3, 48 h after PORV-GD-01-2015 strain infected cells, the TCID 50 The values ​​are 10 -2.125 , 10 -2.25 , while the TCID50 of the empty control group was 10 -4.625 The non-transfected control group was 10 -5.125 It can be seen that the inhibitory effects of pcDNA6.2-VP7-323 and pcDNA6.2-VP6-345 on PORV were significantly different from those of the empty control group and the non-transfected group, and pcDNA6.2-VP7-323 had the best inhibitory effect.

[0096] 3. After 72 hours of infection of Vero cells in step 1, the fixed infected cell plates were subjected to indirect immunofluorescence assay. The results were as follows: Figure 8 to Figure 12 As shown. Figure 8 to Figure 12 It can be seen that the fluorescence intensity of the cells infected with pcDNA6.2-VP7-323 and pcDNA6.2-VP6-345 was significantly lower than that of the empty control group and the non-transfected and infected group, indicating that pcDNA6.2-VP7-323 and pcDNA6.2-VP6-345 can inhibit the replication and proliferation of PORV.

[0097] It can be seen from the above examples that the amiRNA provided by the present invention can effectively inhibit the replication and proliferation of porcine rotavirus PORV-GD-01-2015 strain.

[0098] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An amiRNA that inhibits porcine rotavirus, It is characterized in that The amiRNA includes amiRNA-323 or amiRNA-345; The nucleotide sequence of the target of amiRNA-323 is shown in SEQ ID No.5; The nucleotide sequence of the target of amiRNA-345 is shown in SEQ ID No.

8.

2. The amiRNA according to claim 1, It is characterized in that The nucleotide sequence of the sense strand of the amiRNA-323 is shown in SEQ ID No. 1, and the nucleotide sequence of the antisense strand is shown in SEQ ID No. 2; The nucleotide sequence of the sense strand of the amiRNA-345 is shown in SEQ ID No.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID No.

4.

3. Use of the amiRNA according to claim 1 or 2 in the preparation of a product for preventing and treating porcine rotavirus infection.

4. The use according to claim 3, It is characterized in that The porcine rotavirus includes the PORV-GD-01-2015 strain.

5. The use according to claim 3, It is characterized in that The products include biological products.

6. A product for treating porcine rotavirus infection, It is characterized in that The active ingredient of the product includes the amiRNA according to claim 1.

7. Use of the amiRNA according to claim 1 or 2 in preparing a transgenic cell line resistant to porcine rotavirus infection.

8. Use of the amiRNA according to claim 1 or 2 in breeding transgenic pigs resistant to porcine rotavirus infection for non-therapeutic purposes.

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