Screening and application of anti-feline calicivirus siRNA (small interfering Ribonucleic Acid)
By designing and screening siRNAs against cat calicivirus, the problem that existing treatment methods cannot eradicate the cause is solved, effective silencing of cat calicivirus is achieved, and new treatment plans are provided.
Patent Information
- Application Number
- CN202311858455.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-30
- Publication Date
- 2025-03-18
AI Technical Summary
Currently, there is a lack of effective methods to prevent or treat cat calicivirus infection. The existing treatment methods are mainly symptomatic treatment and cannot eradicate the cause.
By designing and screening siRNAs against cat calicivirus, the silencing effect of siRNA is used to inhibit virus replication. The specific steps include siRNA design, confirmation of transfection efficiency, cytotoxicity exploration and in vitro screening.
A siRNA that can better silence cat calicivirus was screened and verified that it has a good silencing effect on multiple strains, thus providing new possibilities for the treatment of cat calicivirus infection.
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Figure CN119639825A_ABST
Abstract
Description
Technical Field
[0001] The invention provides a method for screening siRNA against feline calicivirus and application thereof. Background Art
[0002] Feline calicivirus infection is a viral respiratory infectious disease of cats, mainly manifested by upper respiratory tract symptoms, namely, depression, serous and mucous rhinorrhea, conjunctivitis, stomatitis, tracheitis, bronchitis, accompanied by biphasic fever. Feline calicivirus infection is a common disease in cats, with high morbidity and low mortality. At present, the main method for treating feline calicivirus infection is symptomatic treatment. Most of the drugs used for symptomatic treatment are used to improve symptoms, or treat the symptoms. Symptomatic treatment cannot eradicate the cause of the disease. There is currently no RNA that interferes with feline calicivirus to prevent or treat feline calicivirus. Therefore, there is an urgent need for a method to screen for anti-feline viruses to solve this problem. Summary of the invention
[0003] In view of the above-mentioned shortcomings, the present invention provides a screening of siRNA against feline calicivirus and its application, which can screen out an siRNA that can effectively silence feline calicivirus, and verify that it has a good silencing effect on multiple strains of feline calicivirus, thereby concluding that the siRNA can be used to treat feline calicivirus infection.
[0004] The present invention adopts the following technical scheme: a screening of siRNA against feline calicivirus, which comprises the following steps: Step 1, siRNA design, first analyze and compare the whole genome sequences of all feline calicivirus strains in GenBank, select the completely conserved regions of all strains, design and synthesize siRNAs from the completely conserved regions; Step 2: Confirm transfection efficiency with fluorescent siRNA. According to the instructions of the transfection reagent, 1.5 × 10 4 - 3.5 × 10 4 cells / 500 μL. When the cell density increased to 30% - 50%, siRNA transfection was performed. Fluorescent siRNA with a concentration of 20 μM was diluted to 50 nM with serum-free cell culture medium, and the diluted fluorescent siRNA and transfection reagent were vortexed to mix evenly. The final volume was 100 μL. The plate was allowed to stand at room temperature for 10 min. The original culture medium in the 24-well plate was discarded, and 500 μL of culture medium containing 10% FBS was added. Then, 100 μL of transfection complex was added after the standing was completed. The plate was gently shaken to mix evenly. Three replicates were set for each group. At the same time, a blank control well without siRNA transfection was set. The plate was placed in a 37 °C incubator for 24 h. The transfection efficiency was observed under a fluorescence microscope. Step 3: To explore the toxicity of siRNA to cells, 2.5 × 10 3 - 7.5 × 10 3 CRFK cells / 100 μL, when the cell density increases to 30% - 50%, siRNA transfection is performed, the universal negative control siRNA with a concentration of 20 μM is diluted, mixed evenly with the transfection reagent, the final volume is 50 μL, and it is allowed to stand at room temperature for 10 min, the original culture medium in the 96-well plate is discarded, 100 μL of culture medium containing 10% FBS is added, and then 50 μL of transfection complex is added, and the mixture is gently shaken. Three replicates are set for each concentration, and a control group without siRNA transfection and a blank group without cells are set at the same time. The plates are placed in a 37 ℃ incubator for 24 h, 10 μL of CCK-8 solution is added to each well, and the mixture is gently shaken. The plates are incubated at 37 ℃ for 1 h, and the absorbance is measured at 450 nm; Step 4: siRNA in vitro screening: 7.5 × 10 3 – 1.7 × 10 4 CRFK cells / 200 μL, when the cell density increased to 30% - 50%, siRNA transfection was performed, the 5 pairs of siRNA in 2.1 were diluted to 50 nM, 1 μL of transfection reagent was added to each and mixed evenly, the final volume was 50 μL, the transfection complex was allowed to stand at room temperature for 10 min, and then added to 48-well plates respectively, and placed in a 37 ℃ incubator for 24 h. At the same time, a control group transfected with negative siRNA and a control group not transfected with siRNA were set up, each with 3 replicates, and the virus titer was 1 × 10 9.3 TCID 50 / mL feline calicivirus HRB23 was inoculated into the siRNA transfection group of a 48-well plate at an MOI of 0.01, and a negative control group without virus infection was set up at the same time. After culturing in a 37°C incubator for 24 h, RNA from all wells was extracted and reverse transcribed. The method was used for qPCR detection and calculation of feline calicivirus nucleic acid copy number.
[0005] Furthermore, the specific information of the siRNA described in step 1 is: .
[0006] Furthermore, the fluorescent siRNA diluted in step 3 was vortexed and mixed evenly with 1 μL, 1.5 μL, 2 μL and 2.5 μL of transfection reagent, respectively.
[0007] Furthermore, in step 4, the universal negative control siRNA with a concentration of 20 μM was diluted to 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, and 400 nM, and mixed evenly with 0.5 μL, 0.625 μL, 0.75 μL, 0.875 μL, 1 μL, 1.125 μL, 1.25 μL, and 1.375 μL of the transfection reagent, respectively.
[0008] The invention also discloses an application of siRNA against feline calicivirus, wherein the siRNA can interfere with feline caliciviruses of different concentrations and different feline calicivirus strains.
[0009] Furthermore, the process of siRNA interference with different concentrations of feline calicivirus was to plate 7.5 × 10 3 – 1.7 × 10 4 CRFK cells / 200 μL, when the cell density increased to 30% - 50%, siRNA transfection was performed, the siRNA with the best silencing effect was diluted to 300 nM, 2.5 μL transfection reagent was added and mixed evenly, the final volume was 50 μL, 5 transfection advantage siRNA groups were set up, each group had 3 replicates, and each group had a control group for transfection of negative siRNA and a control group without transfection. The transfection complex was added to a 48-well plate and placed in a 37 ℃ incubator for 24 h. The feline calicivirus HRB23 strain was inoculated into a 48-well plate with an MOI of 0.001, 0.004, 0.008, 0.01, and 0.04, and a negative control well without virus was set. After 24 h of incubation at 37 ℃, RNA from all wells was extracted and reverse transcribed, and qPCR detection was performed to calculate the number of feline calicivirus nucleic acid copies.
[0010] Furthermore, the process of siRNA interference with different feline calicivirus strains is to transfect the dominant siRNA in a 48-well plate, set up 4 transfection siRNA groups, 3 replicates in each group, and operate the same as 2.5. The virus titers of feline calicivirus F9, feline calicivirus FB-NJ-13, feline calicivirus DL31, and feline calicivirus 2280 strains were determined, and the well plates were inoculated with an MOI of 0.008. After culturing in a 37 °C incubator for 24 h, RNA from all wells was extracted and reverse transcribed, and qPCR detection was performed to calculate the number of feline calicivirus nucleic acid copies.
[0011] The present invention has the following beneficial effects: after adopting the above technical scheme, 5 siRNAs are designed according to the completely conserved regions of all strains of feline calicivirus, and the 5 siRNAs are subjected to the processes of confirming the transfection efficiency, judging the cytotoxicity and in vitro screening, thereby screening out 1 siRNA that can better silence the feline calicivirus, and verifying that it has a good silencing effect on multiple strains of feline calicivirus, thereby concluding that the siRNA can be used for the treatment of feline calicivirus infection. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0013] Figure 1 The transfection efficiency was confirmed by mixing 50 nM fluorescent siRNA of the present invention with 1 μL, 1.5 μL, 2 μL, and 2.5 μL of transfection reagent and then transfecting into CRFK cells (A. transfection reagent 1 μL group; B. transfection reagent 1.5 μL group; C. transfection reagent 2 μL group; D. transfection reagent 2.5 μL group; E. blank control group).
[0014] Figure 2 It is a curve diagram showing the results of the toxicity test of the siRNA of the present invention on CRFK cells.
[0015] Figure 3 This is a schematic diagram comparing the screening results of 5 pairs of siRNA transfected into CRFK cells of the present invention.
[0016] Figure 4 The figure shows the silencing efficiency of siRNA 2.3 of the present invention on feline calicivirus at different concentrations.
[0017] Figure 5 The figure shows the silencing efficiency of siRNA 2.3 of the present invention on different strains of feline calicivirus. DETAILED DESCRIPTION
[0018] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.
[0019] 1. The materials used in the screening process of the present invention are: the transfection reagent is INTERFERin® transfectionreagent from Polyplus, the Cell Counting Kit-8 (CCK-8 kit) is from Bio-Tech, the viral DNA / RNA column extraction kit is from Tiangen Biotech Co., Ltd., the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) is from TaKaRa, 1640 culture medium, FBS ( Fetal bovine serum From Gibco, feline kidney cells (CRFK cells), feline calicivirus SH-1 strain (GenBank: MT239579.1), feline calicivirus HZ-1 strain (GenBank: MT680938.1), and feline calicivirus SX-1 strain (GenBank: MT759583.1) were isolated and stored. Feline calicivirus F9 (GenBank: M86379) and feline calicivirus 2280 (GenBank: KC835209) were from ATCC.
[0020] The present invention provides a screening of siRNA against feline calicivirus, which comprises the following steps: step 1, siRNA design, analyzing and comparing the whole genome sequences of all feline calicivirus strains logged in GenBank, selecting the completely conserved regions of all strains, designing and synthesizing siRNA for the completely conserved regions, and the specific information is shown in Table 1. .
[0021] Step 2: Confirm transfection efficiency with fluorescent siRNA. According to the instructions of the transfection reagent, 1.5 × 10 4 - 3.5 × 10 4 Cells / 500 μL, when the cell density increases to 30% - 50%, siRNA transfection is performed, and the fluorescent siRNA (fluorescent transfection control, Cy3-labeled siRNA) with a concentration of 20 μM is diluted to 50 nM with serum-free cell culture medium, and vortexed and mixed evenly with 1 μL, 1.5 μL, 2 μL, and 2.5 μL of transfection reagent, respectively, to a final volume of 100 μL, and allowed to stand at room temperature for 10 min, the original culture medium in the 24-well plate is discarded, 500 μL of culture medium containing 10% FBS is added, and then 100 μL of transfection complex is added after the standing is completed, and gently shaken to mix evenly. Each group has 3 replicates, and a blank control well without siRNA transfection is set up at the same time, and placed in a 37 ℃ incubator for 24 h, and the transfection efficiency is observed under a fluorescence microscope.
[0022] Step 3: Exploring the cytotoxicity of siRNA to cells. 2.5 × 10 3 - 7.5 × 10 3 CRFK cells / 100 μL, when the cell density increased to 30% - 50%, siRNA transfection was performed, and the universal negative control siRNA with a concentration of 20 μM was diluted to 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, and 400 nM, and mixed evenly with 0.5 μL, 0.625 μL, 0.75 μL, 0.875 μL, 1 μL, 1.125 μL, 1.25 μL, and 1.375 μL of transfection reagent, respectively, with a final volume of 50 μL. The plate was allowed to stand at room temperature for 10 min, the original culture medium in the 96-well plate was discarded, 100 μL of culture medium containing 10% FBS was added, and then 50 μL of transfection complex was added, and the plate was gently shaken to mix. Three replicates were set for each concentration, and a control group without siRNA transfection and a blank group without cells were set up. The cells were placed in a 37°C incubator for 24 h, 10 μL of CCK-8 solution was added to each well, the mixture was gently shaken, and the cells were incubated at 37°C for 1 h. The absorbance was measured at 450 nm.
[0023] Step 4: siRNA in vitro screening process: 7.5 × 10 3 – 1.7 × 10 4 CRFK cells / 200 μL, and when the cell density increases to 30% - 50%, siRNA transfection is performed. Dilute the 5 pairs of siRNA in 2.1 to 50 nM, add 1 μL of transfection reagent to each and mix evenly, the final volume is 50 μL, let the transfection complex stand at room temperature for 10 min, then add them to 48-well plates respectively, and place them in a 37 ℃ incubator for 24 h. At the same time, set up a control group for transfection of negative siRNA and a control group for non-transfection of siRNA, with 3 replicates for each group. The virus titer is 1 × 10 9.3 TCID 50 / mL feline calicivirus HRB23 was inoculated into the siRNA transfection group of a 48-well plate at an MOI of 0.01, and a negative control group without virus infection was set up at the same time. After culturing in a 37 ℃ incubator for 24 h, RNA from all wells was extracted and reverse transcribed. [1] The method performs qPCR detection and calculates the number of feline calicivirus nucleic acid copies.
[0024] 2. The present invention also discloses an application of siRNA against feline calicivirus, which can interfere with feline calicivirus of different concentrations and different strains of feline calicivirus.
[0025] (1) The interference effect of superior siRNA on feline calicivirus at different concentrations was tested by plating 7.5 × 10 3 – 1.7 × 10 4 CRFK cells / 200 μL, when the cell density increased to 30% - 50%, siRNA transfection was performed, the siRNA with the best silencing effect was diluted to 300 nM, 2.5 μL transfection reagent was added and mixed evenly, the final volume was 50 μL, 5 transfection advantage siRNA groups were set up, each group had 3 replicates, and each group had a control group for transfection of negative siRNA and a control group without transfection. The transfection complex was added to a 48-well plate and placed in a 37 ℃ incubator for 24 h. The feline calicivirus HRB23 strain was inoculated into a 48-well plate with an MOI of 0.001, 0.004, 0.008, 0.01, and 0.04, and a negative control well without virus was set. After 24 h of incubation at 37 ℃, RNA from all wells was extracted and reverse transcribed, and qPCR detection was performed to calculate the number of feline calicivirus nucleic acid copies.
[0026] (2) The interference effect of dominant siRNA on different feline calicivirus strains was performed by transfecting dominant siRNA in 48-well plates. Four transfection siRNA groups were set up, with 3 replicates in each group. The operation was the same as in 2.5. The virus titers of feline calicivirus F9, feline calicivirus FB-NJ-13, feline calicivirus DL31, and feline calicivirus 2280 strains were measured, and the virus was inoculated into the well plate at an MOI of 0.008. After culturing in a 37°C incubator for 24 h, RNA from all wells was extracted and reverse transcribed. qPCR detection was performed to calculate the number of feline calicivirus nucleic acid copies.
[0027] 3. Results (1) Confirmation of transfection efficiency by fluorescent siRNA 50 nM fluorescent siRNA was mixed with 1 μL, 1.5 μL, 2 μL, and 2.5 μL of transfection reagent, respectively, and then transfected into CRFK cells. After 24 h, fluorescence microscopy observation showed that 1 μL of transfection reagent could efficiently transfect siRNA, with obvious red fluorescence in the cells, while no cells were seen in the blank control group under red light (see Figure 1 ), indicating that this transfection reagent has high transfection efficiency and this transfection system can be used for subsequent experiments.
[0028] (2) Cytotoxicity test: 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, and 400 nM siRNA were transfected into CRFK cells, and the cell viability was calculated. The results showed that in the 50 nM-300 nM siRNA group, the cells were in good condition and had strong cell viability. When the siRNA concentration was higher than 300 nM, the cell viability was weak and the growth state was poor. Therefore, the maximum siRNA concentration that the cells can withstand is 300 nM, see Figure 2 .
[0029] (3) siRNA in vitro screening: The five pairs of designed siRNAs were transfected into CRFK cells. After 24 hours, the feline calicivirus F9 strain was inoculated with an MOI of 0.2. The qPCR results showed that siRNA 2.3 had the highest silencing efficiency and the largest amount of residual RNA. Figure 3 .
[0030] (4) Interference effect of siRNA 2.3 on feline calicivirus at different concentrations: F9 strains of feline calicivirus with MOIs of 0.01, 0.04, 0.08, 0.2, and 0.4 were inoculated into CRFK cells transfected with 300 nM siRNA 2.3. RNA was extracted 24 hours later and qPCR was performed. The results showed that 300 nM siRNA 2.3 could completely silence feline calicivirus with MOIs of 0.04 and 0.08, see Figure 4 .
[0031] (5) Interference effect of siRNA 2.3 on different strains of feline calicivirus: Feline calicivirus F9, feline calicivirus 2208, feline calicivirus HZ-1, feline calicivirus SH-1 and feline calicivirus SX-1 strains with an MOI of 0.08 were inoculated into CRFK cells transfected with 300 nM siRNA 2.3. RNA was extracted 24 hours later and qPCR was performed. The results showed that siRNA 2.3 had a good silencing effect on different strains of feline calicivirus. This indicates that siRNA 2.3 has a broad spectrum of silencing effects on feline calicivirus strains and has good versatility. Figure 5 .
[0032] Without limitation to this, any changes or substitutions that are not conceived through creative work should be covered within the protection scope of the present invention patent. Therefore, the protection scope of the present invention patent should be based on the protection scope defined in the claims.
Claims
1. Screening of siRNA against feline calicivirus, characterized in that The following steps are involved: Step 1, siRNA design, first analyze and compare the whole genome sequences of all feline calicivirus strains in GenBank, select the completely conserved regions of all strains, design and synthesize siRNAs from the completely conserved regions; Step 2: Confirm transfection efficiency with fluorescent siRNA. According to the instructions of the transfection reagent, 1.5 × 10 4 - 3.5 × 10 4 cells / 500 μL. When the cell density increased to 30% - 50%, siRNA transfection was performed. Fluorescent siRNA with a concentration of 20 μM was diluted to 50 nM with serum-free cell culture medium, and the diluted fluorescent siRNA and transfection reagent were vortexed to mix evenly. The final volume was 100 μL. The plate was allowed to stand at room temperature for 10 min. The original culture medium in the 24-well plate was discarded, and 500 μL of culture medium containing 10% FBS was added. Then, 100 μL of transfection complex was added after the standing was completed. The plate was gently shaken to mix evenly. Three replicates were set for each group. At the same time, a blank control well without siRNA transfection was set. The plate was placed in a 37 °C incubator for 24 h. The transfection efficiency was observed under a fluorescence microscope. Step 3: To explore the toxicity of siRNA to cells, 2.5 × 10 3 - 7.5 × 10 3 CRFK cells / 100 μL, when the cell density increases to 30% - 50%, siRNA transfection is performed, the universal negative control siRNA with a concentration of 20 μM is diluted, mixed evenly with the transfection reagent, the final volume is 50 μL, and it is allowed to stand at room temperature for 10 min, the original culture medium in the 96-well plate is discarded, 100 μL of culture medium containing 10% FBS is added, and then 50 μL of transfection complex is added, and the mixture is gently shaken. Three replicates are set for each concentration, and a control group without siRNA transfection and a blank group without cells are set at the same time. The plates are placed in a 37 ℃ incubator for 24 h, 10 μL of CCK-8 solution is added to each well, and the mixture is gently shaken. The plates are incubated at 37 ℃ for 1 h, and the absorbance is measured at 450 nm; Step 4: siRNA in vitro screening: 7.5 × 10 3 – 1.7 × 10 4 CRFK cells / 200 μL, when the cell density increased to 30% - 50%, siRNA transfection was performed, the 5 pairs of siRNA in 2.1 were diluted to 50 nM, 1 μL of transfection reagent was added to each and mixed evenly, the final volume was 50 μL, the transfection complex was allowed to stand at room temperature for 10 min, and then added to 48-well plates respectively, and placed in a 37 ℃ incubator for 24 h. At the same time, a control group transfected with negative siRNA and a control group not transfected with siRNA were set up, each with 3 replicates, and the virus titer was 1 × 10 9.3 TCID 50 / mL feline calicivirus HRB23 was inoculated into the siRNA transfection group of a 48-well plate at an MOI of 0.01, and a negative control group without virus infection was set up at the same time. After culturing in a 37°C incubator for 24 h, RNA from all wells was extracted and reverse transcribed. The method was used for qPCR detection and calculation of feline calicivirus nucleic acid copy number.
2. The screening of siRNA against feline calicivirus according to claim 1, characterized in that The specific information of siRNA described in step 1 is: 。 3. The screening of siRNA against feline calicivirus according to claim 1, characterized in that The fluorescent siRNA diluted in step 3 was vortexed and mixed evenly with 1 μL, 1.5 μL, 2 μL and 2.5 μL of transfection reagent, respectively.
4. The screening of siRNA against feline calicivirus according to claim 1, characterized in that the steps Fourth, the universal negative control siRNA with a concentration of 20 μM was diluted to 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, and 400 nM, and mixed evenly with 0.5 μL, 0.625 μL, 0.75 μL, 0.875 μL, 1 μL, 1.125 μL, 1.25 μL, and 1.375 μL of transfection reagent, respectively.
5. An application of siRNA against feline calicivirus, characterized in that siRNA can interfere with different concentrations of feline calicivirus and different strains of feline calicivirus.
6. The use of the anti-feline calicivirus siRNA according to claim 5, characterized in that The process of siRNA interfering with different concentrations of feline calicivirus is to lay 7.5 × 10 3 – 1.7 × 10 4 CRFK cells / 200 μL, when the cell density increased to 30% - 50%, siRNA transfection was performed, the siRNA with the best silencing effect was diluted to 300 nM, 2.5 μL transfection reagent was added and mixed evenly, the final volume was 50 μL, 5 transfection advantage siRNA groups were set up, each group had 3 replicates, and each group had a control group for transfection of negative siRNA and a control group without transfection. The transfection complex was added to a 48-well plate and placed in a 37 ℃ incubator for 24 h. The feline calicivirus HRB23 strain was inoculated into a 48-well plate with an MOI of 0.001, 0.004, 0.008, 0.01, and 0.04, and a negative control well without virus was set. After 24 h of incubation at 37 ℃, RNA from all wells was extracted and reverse transcribed, and qPCR detection was performed to calculate the number of feline calicivirus nucleic acid copies.
7. The use of the anti-feline calicivirus siRNA according to claim 5, characterized in that The process of siRNA interference with different feline calicivirus strains is to transfect the dominant siRNA in a 48-well plate, set up 4 transfection siRNA groups, 3 replicates in each group, and operate the same as 2.
5. The virus titers of feline calicivirus F9, feline calicivirus FB-NJ-13, feline calicivirus DL31, and feline calicivirus 2280 strains were determined, and the virus was inoculated into the well plate with an MOI of 0.
008. After culturing in a 37 ℃ incubator for 24 h, the RNA of all wells was extracted and reverse transcribed, and the qPCR detection was performed to calculate the feline calicivirus nucleic acid copy number.