RNA interference sequence for inhibiting immediate early gene ORF121 of carp herpesvirus type II and application thereof

Abstract

The application discloses an RNA interference sequence for inhibiting an immediate early gene ORF121 of a type II carp herpes virus and application thereof, wherein the RNA interference sequence comprises siRNA and / or shRNA, a sense strand sequence of the siRNA is shown as SEQ ID NO: 7, and an antisense strand sequence is shown as SEQ ID NO: 8; a sense strand sequence of the shRNA is shown as SEQ ID NO: 15, and an antisense strand sequence is shown as SEQ ID NO: 16. The siRNA and / or shRNA are designed by targeting different sites of a CDS region of an immediate early gene ORF121 of CyHV-2 for the first time, and experiments prove that the siRNA and / or shRNA can effectively reduce the expression amount of the ORF121 and the virus copy number of a supernatant of a GiCF cell, and the RNAi technology for knocking down the CyHV-2 gene provides a new idea for preparing a vaccine or a medicine against the CyHV-2 virus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and particularly relates to an RNA interference sequence for inhibiting immediate early gene ORF121 of Cyprinid herpesvirus 2 type II and application thereof. BACKGROUND

[0002] Cyprinid herpesvirus 2 (CyHV-2) belongs to the family of Alloherpesviridae and the genus of Cyprinid herpesvirus, and is a large double-stranded DNA enveloped virus that can infect common carp or goldfish and cause herpesvirus hematopoietic necrosis (HVHN). The virus has a genome of about 290 kb, and is composed of at least 140 open reading frames (ORFs) that independently encode functional proteins. Similar to viruses of the Herpesviridae family, the expression of genes of CyHV-2 also presents a time sequence feature. Tang Ruizhe et al. identified 5 immediate early (IE), 34 early (E) and 39 late (L) genes in the CyHV-2 genome by high-throughput sequencing combined with actinomycin D (CHX) and cytarabine (Ara-C) inhibitors (Tang R, Lu L, Wang B, et al. Identification of the immediate-early genes of cyprinid herpesvirus 2 [J]. Viruses, 2020, 12(9): 994.). The IE gene initiates transcription immediately after the virus invades the host and produces transcription activators depending on the host's transcription translation system, which promotes the transcription of E and L genes. The E gene is involved in the replication of viral DNA, and the transcription of the L gene promotes the assembly and release of virions (Gruffat H, Marchione R, Manet E. Herpesvirus late gene expression: a viral-specific pre-initiation complex is key [J]. Frontiers in microbiology, 2016, 7: 869.).

[0003] CyHV-2 was first discovered in Japan in 1992, and then spread to many countries and regions around the world. In 2013, the first case of CyHV-2 infection was found in China (Jung S J, Miyazaki T. Herpesviral haematopoietic necrosis of goldfish, Carassius auratus (L.) [J]. Journal of fish diseases, 1995, 18(3): 211-220; Xu J, Zeng L, Zhang H, et al. Cyprinid herpesvirus 2 infection emerged in cultured gibel carp, Carassius auratus gibelio in China [J]. Veterinary Microbiology, 2013, 166(1-2): 138-144.). CyHV-2 can infect goldfish or goldfish at different growth stages, with high mortality and susceptibility, usually breaking out in spring and autumn, and can cause a series of symptoms such as loss of appetite, lethargy, gill, fin base and body surface bleeding, abdominal swelling, and eventually death in infected fish. Studies have shown that CyHV-2 infection of the host can cause acute infection and establish latency and persistent infection in asymptomatic infected survivors, and persistent infection can be transformed into acute infection under certain conditions, and asymptomatic infected survivors may become an important source of infection (Chai W, Qi L, Zhang Y, et al. Evaluation of Cyprinid Herpesvirus 2 Latency and Reactivation in Carassius gibel [J]. Microorganisms, 2020, 8(3): 445.), the complex and variable infection characteristics make the virus easy to spread, and virus prevention and control is more difficult.

[0004] RNA interference (RNAi) refers to a highly conserved evolutionary phenomenon based on the targeting of homologous mRNA in vivo by exogenous or endogenous double-stranded RNA (dsRNA) molecules and inducing efficient and specific degradation, which widely exists in various organisms and plays an important role in the interaction between hosts and viruses (Downward J. RNA interference [J]. 2004, 328: 1245-1248.). RNA interference is based on the complementarity of short RNA sequences (19-23 nt) and targeted gene sequences and mediates specific silencing, and RNAi molecules include small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA). The mechanism of RNAi-induced gene silencing can be roughly divided into two pathways, one is the classical RNAi pathway, siRNA targets complementary mRNA sequences, and then the Argonaute 2 (Ago2) protein in the RNA-induced silencing complex (RISC) is transiently degraded (Wilson R, Doudna JA. Molecular mechanisms of RNA interference [J]. Annual review of biophysics, 2013, 42: 217.); the other is epigenetic silencing, Argonaute 1 (Ago1) protein enables complementary siRNA to enter the nucleus and target the gene promoter region and form the RNA-induced transcriptional silencing (RITS) complex, inducing heritable and inhibitory epigenetic modifications to silence downstream gene expression (Ahlenstiel C L, Suzuki K, Marks K, et al. Controlling HIV-1: non-coding RNA gene therapy approaches to a functional cure [J]. Frontiers in Immunology, 2015, 6: 474.). A large number of studies have focused on the use of RNAi technology (exogenous delivery of chemically synthesized siRNA or shRNA) to inhibit viral replication in vivo or in vitro for therapy, such as HIV-1, hepatitis B virus (HBV), human papillomavirus (HPV) and human cytomegalovirus (HCMV) (Kelleher A D, Cortez-Jugo C, Cavalieri F, et al. RNAi therapeutics: an antiviral strategy for human infections [J]. Current Opinion in Pharmacology, 2020, 54: 121-129.).

[0005] Currently, RNAi technology is also widely used in the study of anti-aquatic animal viruses, such as white spot syndrome virus (WSSV), grass carp hemorrhagic disease virus (GCRV) and nervous necrosis virus (NNV) (Song H, Sun XY, Kong XH, et al. Application of RNA interference technology in the study of anti-virus and anti-parasite of aquatic animals [J]. Biotechnology Bulletin, 2020, 36(2): 13.). The infection mechanism of CyHV-2 is not clear, and the relevant literature evidence is scarce. The development of vaccines or targeted drugs for the virus is slow and difficult. The traditional method of disinfecting the breeding environment or improving the immunity of the fish has little effect. The spread of CyHV-2 still brings significant economic losses to the crucian carp or goldfish breeding industry. There are few reports on the application of RNAi technology in CyHV-2 replication. Jin Liping et al. designed three siRNAs targeting ORF57 of CyHV-2 and found that the pathogenicity and replication of the virus were significantly inhibited after ORF57 silencing, which provided a reference for the anti-virus treatment and attenuation strain modification of CyHV-2 based on siRNA technology (Jin L, Pan XY, Lin L Y, et al. Effect of siRNA interference on the expression of ORF57 gene of cyprinid herpesvirus type 2 [J]. Acta Hydrobiologica Sinica, 2022, 46(4): 371-378.). In addition, it was found that ORF121 has the highest expression in the tissues of CyHV-2 infected fish and is identified as an IE gene of the virus, indicating that ORF121 may have a regulatory effect on the virus E and L genes or host innate immune genes and affect virus replication (Tang R, Lu L, Wang B, et al. Identification of the immediate-early genes of cyprinid herpesvirus 2 [J]. Viruses, 2020, 12(9): 994; Xu L, Podok P, Xie J, et al. Comparative analysis of differential gene expression in kidney tissues of moribund and surviving crucian carp (Carassius auratus gibelio) in response to cyprinid herpesvirus 2 infection [J]. Archives of virology, 2014, 159(8): 1961-1974.). SUMMARY

[0006] Based on this, the present application first proposes an RNA interference sequence for inhibiting immediate early gene ORF121 of cyprinid herpesvirus type II, which comprises siRNA and / or shRNA.

[0007] The application also provides application of the RNA interference sequence for inhibiting immediate early gene ORF121 of the CyHV-2 in preparation of a reagent for inhibiting expression of the immediate early gene ORF121 of the CyHV-2.

[0008] The application also provides application of the RNA interference sequence for inhibiting immediate early gene ORF121 of the CyHV-2 in preparation of a drug or vaccine against the CyHV-2.

[0009] To achieve the above object, the application adopts the following technical scheme:

[0010] In a first aspect, the application provides the RNA interference sequence for inhibiting immediate early gene ORF121 of the CyHV-2, which comprises siRNA and / or shRNA, wherein:

[0011] the sequence of the sense strand of the siRNA is shown as SEQ ID NO: 7, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO: 8;

[0012] the sequence of the sense strand of the shRNA is shown as SEQ ID NO: 15, and the sequence of the antisense strand of the shRNA is shown as SEQ ID NO: 16.

[0013] The inventors of the application first designed 6 pairs of siRNA and / or shRNA targeting different sites of the CDS region of the immediate early gene ORF121 (Genbank accession number: NC_019495.1) of the CyHV-2, and then delivered the siRNA and / or shRNA after infection of the CyHV-2 in the gynogenetic gibel carp caudal fin cell line (GiCF) or exogenous overexpression of ORF121 in HEK293T cells, and screened the effective knockdown of the target sequence, and finally screened siRNA-424 and shRNA-424:

[0014] The sequence of the siRNA-424 is:

[0015] the sense strand: 5'-AGCUCAUUUCAUUUCAAACTT-3'(SEQ ID NO: 7)

[0016] the antisense strand: 5'-GUUUGAAAUGAAAUGAGCUGC-3'(SEQ ID NO: 8)

[0017] The sequence of the shRNA-424 is:

[0018] the sense strand: 5'-GCAGCTCATTTCATTTCAAACTTCA-3'(SEQ ID NO: 15)

[0019] Antisense strand: 5'-TTCGTCGAGTAAAGTAAAGTTTGAGAG-3' (SEQ ID NO: 16)

[0020] As preferred, the carp herpesvirus type II is a carp herpesvirus type II isolate YC-01.

[0021] In a second aspect, the present application also provides use of the RNA interference sequence for inhibiting immediate early gene ORF121 of carp herpesvirus type II in the preparation of a reagent for inhibiting expression of immediate early gene ORF121 of carp herpesvirus type II, which uses siRNA or shRNA to effectively knock down CyHV-2 immediate early gene ORF121 in GiCF cells.

[0022] In a third aspect, the present application also provides use of the RNA interference sequence for inhibiting immediate early gene ORF121 of carp herpesvirus type II in the preparation of an anti-CyHV-2 drug or vaccine.

[0023] Compared with the prior art, the present application has the beneficial effects that the present application provides RNA interference sequence siRNA (shRNA) targeting CyHV-2 immediate early gene ORF121 for the first time, which can effectively reduce the expression amount of ORF121 and the virus copy number of supernatant of GiCF cells after in vitro delivery, and the use of RNAi technology to knock down CyHV-2 gene provides a new idea for studying the infection and replication mechanism of CyHV-2 and preparing a vaccine or drug for CyHV-2 virus treatment and attenuated strain modification based on the technology. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 4 is the effect of RT-qPCR detection of GiCF cells infected with CyHV-2 and transfected with target siRNA on the expression of ORF121 in the examples.

[0025] Figure 2 Figure 5 is the effect of RT-qPCR detection of GiCF cells infected with CyHV-2 and transfected with target shRNA on the virus copy number of supernatant in the examples.

[0026] Figure 3 Figure 6 is the effect of RT-qPCR detection of HEK293T cells overexpressing ORF121 and transfected with target siRNA on the expression thereof in the examples. DETAILED DESCRIPTION

[0027] The present application will be further described in detail by specific embodiments. The following examples are only used to illustrate the present application, but not used as a limitation on the scope of the present application. In addition, if not specifically stated, the specific technical operation means are the conventional operation steps or conditions of those skilled in the art. If the manufacturer of the reagent or instrument is not stated, it is a conventional commercially available product.

[0028] Example 1

[0029] 1 Material

[0030] 1.1 Experimental strain, cell

[0031] The gyninocypris doyli tail fin cell line (GiCF) was constructed by the laboratory and stored in liquid nitrogen. The culture medium for GiCF cell recovery was M199 medium (purchased from Gibco) containing 10% fetal bovine serum (purchased from Geno Biological) and 1% 100x penicillin-streptomycin (purchased from Gibco), and was cultured in a 27°C constant temperature incubator.

[0032] CyHV-2 isolate YC-01 (isolated by the laboratory and stored at -150°C, hereinafter referred to as CyHV-2) was used to infect cells at 25°C, and after the cells were completely dead, the cell supernatant was collected and filtered with a 0.22μm needle filter to obtain the virus liquid, which was stored at -80°C for standby.

[0033] The culture medium for HEK293T cell recovery was DMEM medium (purchased from cytiva HyClone) containing 10% fetal bovine serum (purchased from Geno Biological) and 1% 100x penicillin-streptomycin (purchased from Gibco), and was cultured in a 37°C, 5% CO2 constant temperature incubator.

[0034] 1.2 Experimental reagent

[0035] Trizol, Lipofectamine 3000 were purchased from Invitrogen company; chloroform was purchased from Shanghai Kelings Reagent Co., Ltd.; anhydrous ethanol, methanol, isopropanol were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; DEPC treated water, 20x PBS, sodium chloride (NaCl), kanamycin, seamless cloning kit were purchased from Shengong Biological (Shanghai) Co., Ltd.; DDH2O, viral genomic DNA / RNA extraction kit (DP315), endotoxin-free plasmid large extraction kit (DP120) were purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd.; Premix Ex Taq TM II (RR820A), PrimeScript TM RT MasterMix (RR036A) were purchased from TaKaRa company; E. coli Trans5a competent cells were purchased from Beijing Quanshi Gold Biological Company; tryptone, yeast extract were purchased from OXOID; agar powder was purchased from Solabio Biological; restriction endonuclease EcoR I, 10x QuickCut Green Buffer, Max DNA Polymerase (R045A), DL2000 DNA Marker were purchased from TaKaRa; YeaRed Nucleic Acid Dye (10000x in water) was purchased from Yixing Biotech Co., Ltd. (Shanghai); Agarose was purchased from Shanghai Lanntuo Biotech.

[0036] 1.3 Experimental instruments

[0037] Vertical clean bench (VD-850, Shanghai Lichenbangxi Instrument Technology Co., Ltd.); PCR instrument (T100TMThermal Cycler, Bio-Rad); desktop high-speed refrigerated centrifuge (H3-16KR, Hunan Kecheng Instrument); ultraviolet spectrophotometer (NanoDrop 2000, Thermo); chemiluminescence imaging system (ChemiDocTM Imaging System, Bio-Rad); fluorescent quantitative PCR instrument (CFX96, Bio-Rad); constant temperature oscillation incubator (INFORS-HT Multitron Standard, Iversen China); vortex shaker (Shanghai Husan Industry); portable high-pressure steam sterilizer (DSX-24L-I, Shanghai Shen'an Medical Instrument Factory).

[0038] 2 Method

[0039] 2.1 Design and synthesis of target siRNA (shRNA)

[0040] The CDS region sequence of CyHV-2 immediate early gene ORF121 (Genbank accession number: NC_019495.1) was searched in NCBI (https: / / www.ncbi.nlm.nih.gov). According to the siRNA design principle, the GC content, the secondary structure of mRNA action site, the base preference requirement of siRNA, etc. were comprehensively considered, and the appropriate siRNA target sequence was found based on the siRNA online design software DSIR (http: / / biodev.extra.cea.fr / DSIR / DSIR.html). Then, the BLAST software on NCBI was used to select the transcript reference sequence database to perform homology analysis on the above siRNA sequence, to exclude the possibility of non-specific inhibition of ORF121 by siRNA, and to preliminarily screen 6 pairs of siRNA sequences, as shown in Table 1. The 6 pairs of siRNA sequences, siRNA-NC and shRNA plasmid vectors pGPU6 / RFP / Neo-ORF121-424 (hereinafter referred to as shRNA-424) and pGPU6 / RFP / Neo-shNC (hereinafter referred to as shRNA-NC) designed in this embodiment were synthesized by Shanghai Jimake Gene Co., Ltd.

[0041] Table 1: siRNA (shRNA) sequences

[0042]

[0043]

[0044] 2.2 CyHV-2 infection of GiCF cells and knockdown of ORF121 by targeting siRNA

[0045] 1) The medium for GiCF cells is M199 medium containing 10% fetal bovine serum and 1% 100x penicillin-streptomycin, and the cells are cultured in a 27°C constant temperature incubator. When the cells grow to the logarithmic phase, they are transferred to a 12-well plate (1-5x10 5 6 cells are inoculated per well). After stable growth (60-80% cell confluence), the GiCF cells are infected with CyHV-2 at an MOI of 0.1 for 2 hours, and then the virus solution is removed, and the cells are ready for transfection;

[0046] 2) Transfection:

[0047] 1 OD of siRNA is resuspended in 125 μL DEPC-treated water to prepare a 20 μM (20 pmol / μL) solution (1 OD duplex ≈ 2.5 nmol ≈ 33 μg);

[0048] The cationic liposome is diluted (63 μL Opti-MEM medium and 1.9 μL Lipofectamine TM 3000 Reagent are added), vortexed and briefly centrifuged; the siRNA is diluted (63 μL Opti-MEM medium and 4 μL siRNA, i.e. 80 pM, are added), vortexed and briefly centrifuged; the siRNA is slowly added to the cationic liposome, mixed gently, incubated at room temperature for 15 min to form a transfection complex;

[0049] Each well is replaced with 700 μL of fresh Opti-MEM medium, and then 125 μL of the transfection complex is added to each well, mixed, and the GiCF cells are cultured in a 27°C incubator for 8 h before extracting the cell RNA for RT-qPCR detection.

[0050] 2.3 CyHV-2 infection of GiCF cells and knockdown of ORF121 by targeting shRNA

[0051] 1) The GiCF cells are cultured to the state described in 2.2 1) and are ready for transfection of the plasmid;

[0052] 2) Transfection:

[0053] shRNA-424 and shRNA-NC plasmids were amplified by transformation of *E. coli* Trans5α competent cells and extracted and purified using an endotoxin-free plasmid large-scale extraction kit. The plasmid DNA concentration and purity were determined by UV spectrophotometry; those meeting the standards were stored at -20℃ for later use. Cationic liposomes were diluted (63 μL Opti-MEM medium, 1.9 μL Lipofectamine) TM 3000 Reagent), vortex and briefly centrifuge; dilute plasmid DNA (add 63 μL Opti-MEM medium, 1000 ng plasmid DNA, 2.5 μL P3000). TM Reagent), vortex and briefly centrifuge; slowly drip plasmid DNA into cationic liposomes, gently mix, incubate at room temperature for 15 min to form transfection complex;

[0054] Replace 700 μL of fresh Opti-MEM medium in each well beforehand, then add 125 μL of the transfection complex to each well and mix well. Incubate GiCF cells at 27°C until they stably express shRNA-424 and shRNA-NC plasmid (vector labeled with RFP fluorescence). Infect with CyHV-2 at MOI=0.1 for 8 h, then remove the virus solution. Collect cell supernatant at 24 h, 48 h, 72 h, and 96 h, and use RT-qPCR to detect the viral copy number in the supernatant (quantitative detection of ORF121).

[0055] 2.4 Overexpression of ORF121 and targeted siRNA knockdown of ORF121 in HEK293T cells

[0056] 1) Construct the pDsRed-express-ORF121 recombinant plasmid:

[0057] The ORF121 fragment was amplified from CyHV-2-infected GiCF cells. The PCR reaction system and conditions were as follows: Max DNA polymerase (Tables 2 and 3) and PCR primers are listed in Table 4. The pDsRed-express-N1 vector (purchased from Clontech) was digested with the restriction endonuclease EcoRI at 37°C for 1 hour. The reaction mixture consisted of 10 μL 10×QuickCut Green Buffer, 2 μg plasmid DNA, 2 μL QuickCut EcoRI, and ddH2O to a final volume of 100 μL. The purified PCR fragment was ligated to the purified linearized vector at 50°C for 30 minutes. The ligation product was transformed into *E. coli* Trans5α competent cells. Single colonies were picked and amplified on plates, and the recombinant plasmid was extracted and sequenced for verification.

[0058] 2) The culture medium for HEK293T cells is DMEM medium containing 10% fetal bovine serum and 1% 100x penicillin-streptomycin, and the cells are cultured in a 37°C, 5% CO2 incubator. When the cells grow to the logarithmic phase, they are transferred to a 12-well plate (1-5x10 5 After stable growth (70% confluence), the pDsRed-express-ORF121 plasmid is transfected for overexpression, and the transfection method is the same as that in 2.3 2) shRNA-424 plasmid vector delivery method. After 24 hours of overexpression, siRNA targeting ORF121 is transfected, and the method is the same as that in 2.2 2) siRNA delivery method. After 8 hours, the cell RNA is extracted for RT-qPCR detection.

[0059] Table 2: PCR reaction system

[0060]

[0061] Table 3: PCR reaction conditions

[0062]

[0063] Table 4: Primers

[0064]

[0065] 2.4 Extraction of total RNA from cells

[0066] The treated cells are collected, 1 mL Trizol is added to each well, and the mixture is blown and mixed. Then 200 μL chloroform is added, and the mixture is vortexed for 15 seconds, allowed to stand for 3 minutes, and centrifuged at 12000xg at 4°C for 15 minutes. The supernatant is taken and an equal amount of isopropanol is added, and the mixture is allowed to stand for 10 minutes and centrifuged at 12000xg at 4°C for 10 minutes. The supernatant is discarded, 1 mL of 75% ethanol (prepared with DEPC-treated water) is added, and the mixture is centrifuged at 7500xg at 4°C for 5 minutes. Then 20 μL of DEPC-treated water is added to dissolve the RNA.

[0067] 2.5 Fluorescent quantitative PCR detection (RT-qPCR)

[0068] The reverse transcription reaction is performed by adding 500 ng of RNA to each 10 μL system, and the PrimeScript TM RT Master Mix Kit is used for the experiment.

[0069] Table 5: Reverse transcription reaction system

[0070]

[0071] Table 6: Reverse transcription reaction conditions

[0072]

[0073] RT-qPCR reaction system refers to TB Green Premix Ex Taq II (Tli RNaseH Plus) Kit. The relative expression of each gene (the internal reference gene of GiCF cells is β-actin, and the internal reference gene of HEK293T cells is GAPDH) is calculated by 2 -ΔΔCt The primers used for RT-qPCR are listed in Table 4.

[0074] Table 7: RT-qPCR reaction system

[0075]

[0076] Table 8: RT-qPCR reaction conditions

[0077]

[0078] 2.6 Detection of viral copy number in cell supernatant

[0079] The recombinant plasmid pDsRed-express-N1-ORF121 was constructed by the laboratory, and the concentration was measured by NanoDrop 2000. The copy number (copies / μL) = 6.02 x 10 23 (copies / mol) x plasmid concentration (ng / μL) x 10 -9 / (recombinant plasmid base number x 660 g / mol), and the plasmid diluted by 10 times was used as the template for RT-qPCR analysis. The cycle number Ct and the logarithm of copy number were used as the horizontal and vertical coordinates to establish the standard curve, respectively. The primers used are listed in Table 4. The viral genomic DNA / RNA extraction kit was used to extract the genomic DNA in 200 μL of viral supernatant, and a total of 30 μL was eluted. The same primers as the standard curve were used for RT-qPCR with viral genomic DNA as the template, and the Ct was converted to viral copy number by the standard curve.

[0080] 3 Results

[0081] 3.1 CyHV-2 infection of GiCF cells and siRNA (shRNA) knockdown of ORF121

[0082] As Figure 1 and 2As shown, siNC (NC): negative control; si145, si237, si303, si424, si687, si777: siRNAs targeting ORF121; shRNA-424: shRNA plasmid targeting ORF121; Note: * and *** indicate p<0.05 and p<0.001 respectively compared with the negative control group (n=3, mean±SEM). GiCF cells were transfected with 6 pairs of siRNAs 2 h after infection with CyHV-2 (MOI=0.1). Compared with the control group, only si424 significantly inhibited the expression of ORF121 at the mRNA level. After stable expression of shRNA-424 in GiCF cells, it significantly inhibited the viral copy number in the cell supernatant 48 h after CyHV-2 infection (MOI=0.1).

[0083] 3.2 Overexpression of ORF121 and targeted siRNA knockdown of ORF121 in HEK293T cells

[0084] like Figure 3 As shown, siNC (NC): negative control; si145, si237, si303, si424, si687, si777: siRNAs targeting ORF121; shRNA-424: shRNA plasmid targeting ORF121; Note: ** indicates p < 0.01 compared to the negative control group (n = 3, mean ± SEM). After HEK293T cells were overexpressed with ORF121 for 24 h, they were transfected with 6 pairs of siRNAs. Compared with the control group, only si424 significantly inhibited the expression of ORF121 at the mRNA level.

[0085] In summary, this invention is the first to discover an siRNA that can inhibit the expression of the CyHV-2 immediate early gene ORF121. After in vitro delivery, it can effectively reduce the expression level of ORF121 and the viral copy number in GiCF cell supernatant. It can be used to prepare vaccines or drugs for CyHV-2 virus treatment or attenuated strain modification.

[0086] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A siRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II, wherein the sequence of the sense strand is shown as SEQ ID NO: 7 and the sequence of the antisense strand is shown as SEQ ID NO:

8.

2. A shRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II, wherein the sequence of the sense strand is shown as SEQ ID NO: 15 and the sequence of the antisense strand is shown as SEQ ID NO:

16.

3. Use of the siRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II according to claim 1 or the shRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II according to claim 2 in the preparation of a medicament for resisting carp herpesvirus type II.

4. Use of the siRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II according to claim 1 or the shRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II according to claim 2 in the preparation of a vaccine for resisting carp herpesvirus type II.

5. A kit for inhibiting or reducing the expression of a gene of carp herpesvirus type II, comprising the siRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II according to claim 1 or the shRNA for inhibiting immediate early gene ORF121 of carp herpesvirus type II according to claim 2.

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