Construction method and application of siRNA for inhibiting expression of canine APN receptor

Abstract

The invention provides a construction method and application of siRNA for inhibiting expression of a canine APN receptor, and belongs to the field of gene engineering. According to the method, siRNA is connected to a saRNA vector. The invention also provides a method for interfering the expression of the canine APN receptor and application of the method in inhibiting canine coronavirus infection. The siRNA is connected with the saRNA vector, expression of APN in mouse blood can be continuously inhibited, and the silencing time is prolonged to 150 days while the silencing effect of the APN in the mouse blood is maintained.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of genetic engineering, and particularly relates to a construction method of siRNA for inhibiting expression of a canine APN receptor and application thereof. BACKGROUND

[0002] Canine coronavirus (CCoV) is a highly contagious enterovirus, and infection with the virus can cause fatal enteritis in puppies, and symptoms such as vomiting, diarrhea, dehydration and the like appear clinically, and dogs of various ages, genders and breeds can be infected, among which the incidence of 2-4 month-old puppies is the highest, and at present, there is no specific antiviral drug, and the prevention of the disease mainly relies on vaccination.

[0003] Coronavirus is related to the presence or absence of aminopeptidase-N (APN) in cells and animal species, and it has been reported that APN is a key host cell receptor of coronavirus, and the expression level directly affects the virus invasion efficiency. However, there is no report on inhibiting the infection of canine coronavirus by blocking the expression of canine APN. SUMMARY

[0004] The application aims to provide a construction method of siRNA for inhibiting expression of a canine APN receptor.

[0005] To achieve the above technical purposes, the application adopts the following technical solutions:

[0006] A construction method of siRNA, wherein the construction method is to connect siRNA to a saRNA carrier, and the

[0007] The nucleotide sequence of the siRNA is shown in SEQ ID NO. 1-SEQ ID NO. 3.

[0008] Further, the elements of the saRNA carrier include a CMV promoter, 5'UTR, nsp1-4, 26S SGP, 3'UTR and Poly(A), wherein the nucleotide sequences of 5'UTR, nsp1-4, 26S SGP, 3'UTR and Poly(A) are shown in SEQ ID NO. 4-SEQ ID NO. 8.

[0009] Further, the connection order of the elements of the saRNA carrier and the siRNA is CMV promoter-5'UTR-nsp1-4

[0010] -26S SGP-siRNA-3'UTR-Poly(A).

[0011] The application also provides application of the construction method of siRNA in interfering with expression of a canine APN receptor.

[0012] The application of the siRNA construction method in the preparation of a drug for inhibiting canine coronavirus infection.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The application first proposes to use siRNA to interfere with the expression of the canine APN receptor.

[0015] The application first proposes to use a method of interfering with the expression of the canine APN receptor to inhibit canine coronavirus infection.

[0016] The application uses a saRNA carrier to connect siRNA, which can continuously inhibit the expression of canine APN, maintains the silencing effect of canine APN, and prolongs the silencing time to 150 days. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 . is a CCK8 detection trend chart at OD450nm;

[0018] Figure 2 . is a Western Bolt detection result chart of three saAPN transfected MDCK cells using APN antibodies;

[0019] Figure 3 . is a result chart of APN expression at different times;

[0020] Figure 4 . is a chart of reverse transcription level expression changes of three saAPN transfected MDCK cells using APN primers;

[0021] Figure 5 . is a reverse transcription level detection result chart after virus infection;

[0022] Figure 6 . is a CCoV fluorescent antibody detection result chart, wherein A is the immunofluorescence result of CCoV epidemic strain infected MDCK cells, B is the immunofluorescence result of saAPN transfected MDCK cells infected with CCoV epidemic strain, and C is the immunofluorescence result of saAPN transfected MDCK cells;

[0023] Figure 7 . is an imageJ immunofluorescence intensity analysis result chart;

[0024] Figure 8 . is a concentration result chart of APN at different times. DETAILED DESCRIPTION

[0025] The following specific descriptions are exemplary and are intended to further explain the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0026] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified.

[0027] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

[0028] Example 1: siRNA design, assembly and in vitro verification

[0029] 1. Experimental materials

[0030] MDCK cells were provided by Ripp Biological Co., Ltd. siRNA synthesis was provided by Beijing Chengke Biological Technology Co., Ltd. Lipofectamine 3000 was purchased from Thermo Fisher. DMEM + 10% FBS + 1% penicillin-streptomycin was purchased from Gibco. RIPA lysis buffer, BCA protein quantification kit, and APN antibody were purchased from Abeam.

[0031] 2. Experimental methods

[0032] 2.1 Design and synthesis of siRNA

[0033] The mRNA sequence of the APN gene is from NM_001003142.1. Using Dharmacon siDESIGN Center, the design region is located, and sequences in the 650-1500 nt interval are selected to design 4 candidate siRNAs.

[0034] Specificity verification: using NCBI BLASTn to input the antisense sequence of the candidate siRNA.

[0035] Off-target effect prediction: using the siRNAscanner tool to scan the dog genome for possible cross-silencing genes.

[0036] Three optimal candidate siRNAs were screened out: siRNA1, siRNA2, and siRNA3, the nucleic acid sequences of which are shown in SEQ ID NO. 1-SEQ ID NO. 3.

[0037] siRNA-1: position: 873-893 nt, GC = 47.6%, 5' end = G

[0038] siRNA-2: Position: 1021-1041 nt, GC=42.9%, 5' end=C

[0039] siRNA-3: Position: 1356-1376 nt, GC=38.1%, 5' end=G

[0040] The nucleic acid sequence was synthesized by Beijing Qikexin Biotechnology Co., Ltd. The 3' end dTdT overhang was 2'-O-methyl modified at a specific position. The synthesis report purity was >98%.

[0041] 2.2 saRNA vector design and construction

[0042] 5'UTR: 44nt 5'UTR of VEEV TC-83 strain (GenBank: NC_075022.1) was used, G3A mutation, GC content 30%-60%.

[0043] nsp1-4: nsp1 (535aa methyltransferase) nsp2 (794aa protease), nsp3 / 4 (replication complex core), nsp2 Q739L, retaining 51nt CSE of nsp1.

[0044] 26S promoter (26S SGP): 26S of SFV was selected, 3'UTR: VEEV wild type,

[0045] Poly(A): Design more than 50nt adenosine sequence.

[0046] The sequence diagram of the elements of the saRNA vector connected to the siRNA: CMV promoter→5'UTR→nsp1-4→26S SGP→siRNA1-siRNA3→3'UTR→Poly(A). Codon optimization was performed using GENScript, and the GC content was controlled.

[0047] The nucleotide sequences encoding 5'UTR, nsp1-4, 26S SGP, 3'UTR, and Poly(A) are shown in SEQ ID NO. 4-SEQ ID NO. 8.

[0048] 2.3 Assembly cloning

[0049] Restriction enzyme EcoRl linearization vector was used for ligation, Gibson assembly was used, and the company was commissioned for synthesis.

[0050] In vitro transcription: Linearized plasmid + T7 RNA polymerase to generate saRNA, DNase 1 digestion to remove residual DNA.

[0051] 2.4, saRNA in vitro verification

[0052] 2.4.1 Cell preparation: MDCK cells were seeded in 12-well plates at a density of 1 x 10 5 cells per well. Incubate at 37°C for 24 h. The cell density reached 70-80%.

[0053] 2.4.2 Transfection complex preparation: Take 1.5 μL saRNA + 50 μL Opti-MEM serum-free medium into tube A. The negative control is Non-targeting saRNA. Take 2 μL Lipofectamine 3000 + 50 μL Opti-MEM into tube B. Gently mix tube A with tube B, and stand at room temperature for 15 min to form saRNA-lipid complex.

[0054] 2.4.3 Cell transfection: Discard the culture medium in the 12-well plate, and add 500 μL serum-free DMEM per well, and add 100 μL saRNA-lipid complex per well. Incubate at 37°C for 6 h, and then replace with complete medium containing 2% FBS. Continue to incubate for 96 h, and observe the cell morphology every 24 h and perform CCK8 detection, OD450nm, and relative cell survival rate calculation. The results are shown in Table 1. The trend chart is shown in Figure 1 , and the results show that there is no obvious difference under the microscope after transfection of saRNA, and there is no significant difference between the experimental group and the blank control group (p>0.05), which proves that the transfection of the three saRNAs has no significant effect on the cells, and subsequent verification experiments can be performed.

[0055] Table 1 is the CCK8 detection OD 450 and relative cell survival rate calculation results at different times

[0056]

[0057] 2.4.4 Protein level APN detection:

[0058] Aspirate the culture medium, and wash twice with PBS. Add 100 μL RIPA lysis buffer per well, and lyse on ice for 30 min. Determine the protein concentration using the BCA method. At the same time, use 12% SDS-PAGE to run the gel, transfer the membrane, and block with 5% skim milk for 1 h. Incubate the APN primary antibody (1:1000) at 4°C overnight, and then incubate the HRP secondary antibody (1:5000) at room temperature for 1 h before developing. The results are shown in Figure 2 . Quantify the gray value using ImageJ, and the results are shown in Figure 3 .

[0059] 2.4.5 Reverse transcription level detection:

[0060] Extract total RNA by TRIzol method, treat with DNase I, synthesize cDNA by reverse transcription, perform qPCR reaction, and calculate the relative expression of APN gene, with GAPDH as the internal reference. The results are shown inFigure 3 The results showed that the expression level of saRNA in the group was significantly lower than that in the control group (p<0.05), proving that the three designed saRNAs could significantly inhibit the expression of APN protein.

[0061] Example 2: Verification of Viral Infection and APN Suppression

[0062] 1. Test materials

[0063] MDCK cells transfected for 48 hours and canine coronavirus isolates A and B were provided by Ruipu Biotechnology Co., Ltd. Maintenance medium: DMEM + 2% FBS was purchased from Gibco. CcoV-N protein antibody was purchased from Santa. CcoV-N gene primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0064] 2. Test Procedure

[0065] 2.1 Virus activation and titration

[0066] Take 100 μL of CcoV strain thawed on ice 24 hours before infection, and dilute to 10 with 900 μL of maintenance solution. -1 Concentration. Diluted virus was inoculated into MDCK cells, and TCID was calculated by immunofluorescence after 72 hours. 50 The results are shown in Table 2. The viral titers of the experimental groups (saRNA-1, saRNA-2, and saRNA-3) were 2.75, 2.63, and 2.92 TCID, respectively. 50 / mL, the titer of the control group (Non-targeting siRNA) was 7.17 TCID. 50 / mL. The titer in the blank control group was 7.08 TCID. 50 / mL. There was no significant difference between the control group and the blank control group (p<0.05). The experimental group showed significant differences compared to the control group and the blank control group (p<0.05), demonstrating that MDCK cells transfected with saRNA lacked a large number of APN target sites, preventing the virus from successfully invading and infecting the cells, thus proving its significant inhibitory effect on the virus. See the trend results for details. Figure 4 Using GAPDH as an internal control, the reverse transcription level in the saRNA group was significantly lower than that in the control group within 96 hours (p<0.05), demonstrating that the three saRNAs can significantly inhibit the synthesis and expression of APN in vitro without having a significant effect on cells.

[0067] Table 2. TCID detection in saRNA-MDCK cells 72 hours after CCoV virus infection. 50

[0068] Sample name lg TCID50 / mL saRNA-1 2.75±0.20 saRNA-2 2.63±0.10 saRNA-3 2.92±0.21 Control group 7.17±0.12 Blank group 7.08±0.12

[0069] 2.2 Cell Preparation

[0070] Day 0: MDCK cells were plated (12-well plate, 1×10⁵ / well). Experimental group (saRNA-1, saRNA-2, saRNA-3), control group (Non-targeting saRNA), blank control group.

[0071] Day 1: Transfection with saRNA (100 nM)

[0072] Day 3: Western blot confirmed APN expression inhibition.

[0073] 2.3 Viral infection

[0074] Aspirate the culture medium from the 12-well plates, wash twice with PBS, add 500 μL of virus dilution buffer (MOI = 1) to the experimental and control groups, and add an equal volume of virus-free maintenance medium to the blank control group. Incubate at 37°C for 2 hours, gently shaking the plate every 15 minutes to ensure even virus coverage. After 2 hours, remove any unadsorbed virus and wash three times with PBS. Add 1 mL of maintenance medium to each well. Continue incubation at 37°C for 72 hours.

[0075] 2.4 Sample Collection and Testing

[0076] 2.4.1 Take 200 μL of supernatant and perform 10-fold serial dilutions. Seed the cells in 96-well plates with 6 wells per dilution, using 100 μL of supernatant + 100 μL of MDCK cells (2 × 10⁴ / well) per well. After 72 h, record the number of CPE-positive wells and calculate TCID. 50 See results Figure 5 The relative expression level of the viral N gene was significantly lower than that of the control group (p < 0.05). This demonstrates that the saRNA inhibits the transcription stage of the cellular APN gene, preventing protein synthesis and thus blocking CCoV invasion.

[0077] 2.4.2 qPCR detection of viral RNA replication

[0078] Total RNA was extracted using the TRIzol method, treated with DNase I, and reverse transcribed to synthesize cDNA. qPCR was then performed to calculate the relative expression level of the viral N gene, using GAPDH as an internal control. Results are shown below. Figure 6 .

[0079] 2.4.3 Immunofluorescence detection of viral proteins

[0080] Fixed with 4% paraformaldehyde for 15 min, then permeabilized with 0.1% Trion X-100 for 10 min. Incubated overnight at 4°C with primary antibody against CCoV-N protein (1:200), followed by incubation at room temperature for 1 h with secondary antibody against Alexa Fluor 594 (1:500). The percentage of positive cells was counted using fluorescence microscopy; results are shown below.Figure 7 The expression level of CCoV-N protein in the saRNA group was not significantly different from that in the negative control (p < 0.05), but was significantly different from that in the positive control group (p < 0.05).

[0081] 2.4.4 Validation of persistence in mice

[0082] To verify the persistence of the saRNA-lipid complex, we immunized 15 C57BL / 6 mice via oral and intramuscular injection. Five mice were in each of the siRNA-lipid complex immunization group and the saRNA-lipid complex immunization group. Blood samples were collected from the buccal vein before immunization and at 10, 20, 30, 50, 70, 100, and 150 days after immunization. Serum was separated, and APN expression was detected by ELISA. The results are shown below. Figure 8 The results showed that the inhibitory effect of the saRNA group lasted for 150 days, and no abnormal behavior was observed in the mice. The inhibitory effect of the siRNA group rebounded after 30 days and returned to the normal serum APN expression level at 100 days. This demonstrates that the designed saRNA can inhibit APN expression for a longer period of time.

[0083] In summary, saRNA blocks APN transcription and prevents its protein synthesis without affecting normal cellular function. It also showed significant inhibitory effects in experiments with prevalent CCoV strains. In mice, saRNA exhibited a more persistent silencing effect compared to siRNA, suggesting this design could be used as a preventative measure against CCoV for early immunization and longer-lasting protection.

[0084] The above embodiments are merely illustrative and not restrictive. The scope of the invention is defined by the claims. Those skilled in the art will understand that various modifications and variations can be made to the invention without departing from the spirit and scope of this application, and all such modifications and variations are within the scope of the invention.

[0085] SEQ ID NO.1: 5'-GCATCAAGTTCGTGGACATdTdT-3'

[0086] SEQ ID NO.2: 5'-CCTGGACATCATCGAGAAAdTdT-3'

[0087] SEQ ID NO.3: 5'-GGATCCAGTACATGTACAAdTdT-3'

[0088] SEQ ID NO.4: 5'-CUAAUCCGGCUCAUAGCCGUUGUUGACAGCGACACCGUGCUUCUAGCUGCGAGGAAGCGGCGG-3'

[0089]

[0090] SEQ ID NO.6:5'-CGGGACTATCTCTTGCTAACCCTAAAGGG-3'

[0091] SEQ ID NO.7:5'-GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCTGCAG-3'

[0092] SEQ ID NO.8:5'-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUACGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCC-3'

Claims

1. A method for constructing siRNA, characterized in that, The construction method involves linking siRNA to a saRNA vector, and the nucleotide sequence encoding the siRNA is shown in SEQ ID NO.1 to SEQ ID NO.

3.

2. The method for constructing siRNA according to claim 1, characterized in that, The elements of the saRNA vector include: The CMV promoter, 5'UTR, nsp1-4, 26S SGP, 3'UTR, and Poly(A) are shown in SEQ ID NO.4 to SEQ ID NO.

8.

3. The method for constructing siRNA according to claim 1, characterized in that, The linking sequence of the saRNA vector element and siRNA is CMV promoter—5'UTR—nsp1-4—26S SGP—siRNA—3'UTR—Poly(A).

4. The application of the siRNA construction method of claim 1 in interfering with canine APN receptor expression.

5. The application of the method for constructing the siRNA according to claim 1 in the preparation of drugs for inhibiting canine coronavirus infection.

Images