SiRNA for targeted inhibition of large yellow croaker SOCS1 gene expression and application thereof

Abstract

The invention discloses siRNA for inhibiting expression of a large yellow croaker SOCS1 gene and application of the siRNA. The target gene cDNA sequence of the siRNA is as shown in SEQ ID NO.1. By transfecting siRNA (siSOCS1-237) into a large yellow croaker head kidney cell strain, the expression of SOCS1 in the cell can be specifically inhibited, and the expression of an anti-virus immune gene of the large yellow croaker is promoted, so that the anti-virus immune response of the large yellow croaker is enhanced. The invention proves that the siRNA is expected to be developed into a novel pseudosciaena crocea antiviral drug, and lays a foundation for preventing and controlling marine fish virus diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acids, and particularly relates to an siRNA for inhibiting the expression of the SOCS1 gene of large yellow croaker and its application. Background Art

[0002] Suppressors of cytokine signaling 1 (SOCS1) is one of the negative regulators of type I interferon (IFN) signaling, and inhibits the antiviral immune response by negatively regulating the JAK-STAT signaling pathway mediated by type I IFN. Existing studies have shown that viruses such as respiratory syncytial virus and flavivirus utilize SOCS1 to negatively regulate the antiviral immune response of the host (Seong R, et al 2020. Zika Virus-Induction of the Suppressor of Cytokine Signaling 1 / 3 Contributes to the Modulation of Viral Replication. Pathogens 9.). SOCS1 of fish such as Sebastes Schlegeli Sebastes schlegelii Miichthys miiuy also plays a negative regulatory role in antiviral immunity (Wang G, et al. 2022. Molecular characterization and immunoregulatory analysis of suppressors of cytokine signaling 1 (SOCS1) in black rockfish, Sebastes Schlegeli . Dev Comp Immunol. 130: 104355; Zhao X, et al. 2018. Characterization and role of suppressor of cytokine signaling 1a (SOCS1a) in a teleost fish, Miichthys miiuy . Dev Comp Immunol. 78:124-131.). Given the negative regulatory role of SOCS1 in the antiviral immune response, it can be used as a target for the antiviral immune response.

[0003] RNA interference technology is a gene silencing technology triggered by endogenous or exogenous double-stranded RNA (dsRNA). Specifically, this technology uses double-stranded RNA to specifically bind to homologous mRNA in cells, thereby inducing the degradation of target mRNA, thereby preventing its translation process and achieving the purpose of inhibiting the expression of target genes. RNA interference technology can accurately regulate the expression of target genes, with strong specificity and high inhibition efficiency. At the same time, RNA interference technology is simple and easy to use, simple to synthesize, and widely used in disease treatment, drug screening and other fields.

[0004] So far, RNA interference technology has not been used to regulate the expression of SOCS1 gene in large yellow croaker and its antiviral immune response. Summary of the invention

[0005] The purpose of the present invention is to provide a siRNA for targeting and inhibiting the expression of SOCS1 gene in large yellow croaker and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: A siRNA for inhibiting the expression of SOCS1 gene of large yellow croaker by targeting, wherein the siRNA is siSOCS1-237, the sense strand sequence of siSOCS1-237 is: 5'-GAACCCGAGAUCAGUGUCU dTdT-3', and the antisense strand sequence is: 5'-AGACACUGAUCUCGGGUUCdTdT-3'; the nucleotide sequence of the target gene of the siRNA is shown in SEQ ID NO.1.

[0007] The step of siSOCS1-237 targeted inhibition of large yellow croaker SOCS1 gene expression is to transfect siSOCS1-237 into large yellow croaker head kidney cells, causing the mRNA level of the target gene SOCS1 to decrease, thereby inhibiting the expression of large yellow croaker SOCS1.

[0008] An application of the above-mentioned siRNA that targets and inhibits the expression of SOCS1 gene in large yellow croaker to promote the expression of antiviral immune genes in large yellow croaker.

[0009] The large yellow croaker antiviral immunity genes include type I interferon IFNi, PKR, Viperin and MxA.

[0010] A use of the above-mentioned siRNA that targets and inhibits the expression of SOCS1 gene in large yellow croaker antiviral drugs.

[0011] The beneficial effects of the present invention are: The present invention provides a siRNA (siSOCS1-237) that can target large yellow croaker SOCS1, which can be transferred into large yellow croaker head kidney cell line to effectively reduce the mRNA level of SOCS1, with an inhibition efficiency of 45%. At the same time, it can also enhance the transcription level of type I interferon (IFNi) and antiviral immune genes (PKR, Viperin, MxA), thereby enhancing the antiviral immune response of large yellow croaker, indicating that siSOCS1-237 can be used for the development of antiviral drugs for large yellow croaker. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the result of siRNA inhibiting the expression level of SOCS1 gene in large yellow croaker.

[0013] Figure 2 The expression changes of type I interferon IFNi in yellow croaker head kidney cells at 12, 24 and 48 hours after siRNA transfection.

[0014] Figure 3 The expression changes of antiviral genes PKR, Viperin and MxA in yellow croaker head kidney cells at 12, 24 and 48 hours after siRNA transfection. DETAILED DESCRIPTION

[0015] In order to make the technical solutions and advantages of the present invention more clear, the invention contents of the present invention are described in detail through the following examples, but they should not be regarded as limiting the scope of the present invention.

[0016] The methods used in the implementation of the following cases are conventional methods unless otherwise specified, and the materials used are commercially available unless otherwise specified.

[0017] Example 1 Design and synthesis of siRNA sequences targeting SOCS1 of large yellow croaker According to the basic principles of siRNA design, a siRNA (siSOCS1-237) was screened from the cDNA sequence of the SOCS1 gene of large yellow croaker (GenBank accession number: XM_010729149.3). The sense strand sequence of the siRNA was 5'-GAACCCGAGAUCAGUGUCUdTdT-3' (SEQ ID NO. 2), and the antisense strand sequence was 5'-AGACACUGAUCGGGUUCdTdT-3' (SEQ ID NO. 3). Two dTdTs were added to the 3' end of siSOCS1-237 to enhance the stability of the siRNA double-stranded complex. The siSOCS1-237 sequence was searched by BLAST in the large yellow croaker genome database of GenBank, and sequences with more than 3 base mismatches with non-homologous genes were selected to exclude the possibility of nonspecific inhibition. Subsequently, the siRNA was synthesized by Beijing Qingke Company, and a negative control siRNA (si-NC) was synthesized at the same time. The sense strand sequence of si-NC was: 5'-UUCUCCGAACGUGUCACGUdTdT-3', and the antisense strand sequence was: 5'- ACGUGACACGUUCGGAGAA dTdT-3'.

[0018] Example 2 Effect of siSOCS1-237 on SOCS1 expression in large yellow croaker head kidney cell line (1) Transfection of siSOCS1-237: Yellow croaker kidney cell line (LYCK) was established in our laboratory and deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: C2013113. LYCK cells were cultured at 1×10 6 / well inoculated into 6-well plates, added 2 mL of prepared M199 complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin from Gibco, USA), and cultured in an incubator at 28°C and 5% carbon dioxide. When the cells grew to 60-80%, the synthesized siRNA was transfected into LYCK using Lipofectamine 2000 (Invitrogen), according to 5 μL: 200pmoL, and the final concentration of siRNA was 100 nM / well. 24 h after transfection, the total RNA of the cells was extracted using an RNA extraction kit (RC20; Novozymes), and then the RNA was reverse transcribed into cDNA using an RNA reverse transcription kit (A5001; Promega).

[0019] (2) Detection of SOCS1 expression: The reverse transcribed cDNA was used as a template for fluorescence quantitative PCR reaction. The 20 μL reaction system was as follows: 1 μL cDNA template, 0.5 μL each of 10 μM upstream and downstream primers, 10 μL SYBR Green Mix (Novozymes), and 8 μL sterile water; reaction procedure: 95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 60°C annealing and extension for 30 s, 40 cycles; 60-95°C melting curve analysis for 10 min. Actin was used as an internal reference in the fluorescence quantitative PCR reaction. The primer sequences are shown in Table 1. The relative expression level of SOCS1 in large yellow croaker was analyzed. The results are shown in Table 1. Figure 1 As shown, after transfection with siSOCS1-237, the mRNA level of SOCS1 in large yellow croaker LYCK cells decreased significantly, with a decrease of about 45%, indicating that siSOCS1-237 significantly inhibited the expression of large yellow croaker SOCS1.

[0020] Table 1 Primers used in fluorescence quantitative PCR experiments

[0021] Example 3 Effect of siSOCS1-237 on type I interferon IFNi and antiviral gene expression in large yellow croaker head kidney cell line The synthesized siRNA was transfected into LYCK cells, and the transfection process was the same as step (1) of Example 2. The cells were collected 12 h, 24 h, and 48 h after transfection, and the total cell RNA was extracted according to the steps of the RNA extraction kit, and then the reverse transcription reaction was performed according to the steps of the RNA reverse transcription kit. The expression changes of interferon gene (IFNi) and antiviral genes (PKR, Viperin and MxA) in LYCK cells were analyzed by the fluorescent quantitative PCR method in Example 2, and the primers are shown in Table 1. The results are shown in Figure 2 and Figure 3 As shown in the figure, after transfection with siSOCS1-237, the expression levels of type I interferon IFNi and antiviral genes PKR, Viperin and MxA in large yellow croaker LYCK cells increased with the interference time, among which the expression levels of IFNi and MxA reached the highest values ​​at 24 h, and the expression levels of PKR and Viperin reached the highest values ​​at 48 h. The experimental results showed that siSOCS1-237 can promote the expression of large yellow croaker interferon gene IFNi and antiviral genes (PKR, Viperin and MxA) and enhance the antiviral immune response.

[0022] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A siRNA targeting and inhibiting the expression of SOCS1 gene in large yellow croaker, characterized in that: The siRNA is siSOCS1-237, the sense strand sequence of siSOCS1-237 is: 5'-GAACCCGAGAUCAGUGUCU dTdT-3', and the antisense strand sequence is: 5'-AGACACUGUCUCGGGUUC dTdT-3'; the nucleotide sequence of the target gene of the siRNA is shown in SEQ ID NO.

1.

2. The siRNA targeting and inhibiting the expression of SOCS1 gene of large yellow croaker according to claim 1, characterized in that: The step of siSOCS1-237 targeted inhibition of large yellow croaker SOCS1 gene expression is to transfect siSOCS1-237 into large yellow croaker head kidney cells, causing the mRNA level of the target gene SOCS1 to decrease, thereby inhibiting the expression of large yellow croaker SOCS1.

3. Use of the siRNA targeting and inhibiting the expression of SOCS1 gene in large yellow croaker as claimed in claim 1 in promoting the expression of antiviral immune genes in large yellow croaker.

4. The use according to claim 3, characterized in that: The large yellow croaker antiviral immunity genes include type I interferon IFNi, PKR, Viperin and MxA.

5. Use of the siRNA targeting and inhibiting the expression of SOCS1 gene of large yellow croaker as claimed in claim 1 in large yellow croaker antiviral drugs.

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