Use of ssc-miR-148a-5p in preparation of anti-pdcoV proliferation drugs and vaccines

Abstract

The application discloses application of ssc-miR-148a-5p in preparation of anti-PDCoV proliferation drugs and vaccines, and belongs to the technical field of antiviral drugs.The application respectively transfects a mimic for increasing expression of ssc-miR-148a-5p and an inhibitor for reducing expression of ssc-miR-148a-5p into pig small intestinal epithelial cells (IPEC-J2), uses PDCoV to infect the cells 24 hours after the transfection, detects the expression amount of PDCoV M gene in the cells by using a fluorescent quantitative PCR technique 24 hours after the infection of the virus, finds that the expression amount of PDCoV M gene is significantly reduced and increased respectively, and indicates that ssc-miR-148a-5p can regulate the proliferation of PDCoV in IPEC-J2 cells.The application provides a new candidate miRNA for preparation of anti-PDCoV proliferation drugs and vaccines.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug technology, and more specifically to the application of ssc-miR-148a-5p in the preparation of anti-PDCoV proliferation drugs and vaccines. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) is a pathogenic enteropathogenic diarrheal virus in pigs, causing watery diarrhea, vomiting, dehydration, and death. Currently, there are no commercially available vaccines or specific drugs for the prevention and treatment of porcine diarrhea caused by this virus, highlighting the urgent need to develop new antiviral strategies.

[0003] MicroRNAs (miRNAs / miRs) are members of the non-coding RNA family that regulate the expression of functional genes at the post-transcriptional level and are widely involved in the regulation of various biological processes. Studies have found that avian miR-148a-5p (gga-miR-148a-5p) can significantly regulate the replication of avian leukosis virus subgroup J (ALV-J) and duck Tembusu virus (DTMUV), but it is unclear whether porcine miR-148a-5p can regulate the proliferation of susceptible porcine viruses.

[0004] This invention discovers that porcine miR-148a-5p (ssc-miR-148a-5p) can regulate the proliferation of PDCoV in porcine small intestinal epithelial cells (IPEC-J2). This invention provides a new molecular target for the preparation of anti-PDCoV proliferation drugs and vaccines. Summary of the Invention

[0005] In view of this, the present invention provides the application of ssc-miR-148a-5p in the preparation of anti-PDCoV proliferation drugs and vaccines.

[0006] This invention transfected porcine small intestinal epithelial cells (IPEC-J2) with a mimic that increases ssc-miR-148a-5p expression (nucleotide sequence shown in SEQ ID NO.1) and an inhibitor that decreases ssc-miR-148a-5p expression (nucleotide sequence shown in SEQ ID NO.2), respectively. Twenty-four hours after transfection, the cells were infected with the PDCoV-TJ1 strain. Quantitative real-time PCR was used to detect the expression level of the PDCoV M gene in the cells after infection. The results showed that the expression levels of the PDCoV M gene were significantly decreased and increased, respectively, indicating that ssc-miR-148a-5p can inhibit the proliferation of PDCoV in IPEC-J2 cells. This invention provides candidate miRNAs for the preparation of anti-PDCoV proliferation drugs and vaccines.

[0007] AAAGUUCUGAGACACUCCGACU, SEQ ID NO.1;

[0008] AGTCGGAGTGTCTCAGAACTTT, SEQ ID NO. 2.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] Application of ssc-miR-148a-5p in the preparation of anti-PDCoV proliferation drugs and vaccines;

[0011] The nucleotide sequence of the ssc-miR-148a-5p is shown in SEQ ID NO.1;

[0012] AAAGUUCUGAGACACUCCGACU, SEQ ID NO.1.

[0013] The above-mentioned ssc-miR-148a-5p is used as a target in the preparation of drugs and / or vaccines for the treatment and / or prevention of PDCoV.

[0014] Furthermore, it is necessary to increase the expression level of ssc-miR-148a-5p.

[0015] A drug to inhibit PDCoV proliferation includes substances that increase the expression of ssc-miR-148a-5p.

[0016] Furthermore, the nucleotide sequence of the substance that increases ssc-miR-148a-5p expression is as follows:

[0017] AAAGUUCUGAGACACUCCGACU, SEQ ID NO.1.

[0018] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention transfects a mimic that increases ssc-miR-148a-5p expression and an inhibitor that decreases ssc-miR-148a-5p expression into porcine small intestinal epithelial cells (IPEC-J2), respectively. 24 hours after transfection, the cells are infected with PDCoV. 24 hours after infection, the expression level of the PDCoV M gene in the cells is detected by real-time quantitative PCR. It was found that the expression level of the PDCoV M gene was significantly reduced and increased, respectively, indicating that ssc-miR-148a-5p can regulate the proliferation of PDCoV in IPEC-J2 cells. The present invention provides a new candidate miRNA for the preparation of anti-PDCoV proliferation drugs and vaccines. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 This is the result of PDCoV M gene expression level after PDCoV infection of IPEC-J2 cells in Example 1 of the present invention;

[0021] Figure 2 The relative expression level of ssc-miR-148a-5p after PDCoV infection of IPEC-J2 cells in Example 2 of this invention is given, where *** represents P<0.001;

[0022] Figure 3 To increase the expression of ssc-miR-148a-5p in PDCoV-infected IPEC-J2 cells for the ssc-miR-148a-5p mimic in Example 3 of this invention, where *** represents P<0.001;

[0023] Figure 4 In Example 3 of this invention, increasing the expression of ssc-miR-148a-5p can inhibit the proliferation of PDCoV in IPEC-J2 cells, where *** represents P<0.001;

[0024] Figure 5 The ssc-miR-148a-5p inhibitor in Example 4 of this invention reduces the expression of ssc-miR-148a-5p in PDCoV-infected IPEC-J2 cells, where *** represents P<0.001;

[0025] Figure 6 In Example 4 of this invention, reducing the expression of ssc-miR-148a-5p can promote the proliferation of PDCoV in IPEC-J2 cells, where *** represents P<0.001. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] IPEC-J2 cells were purchased from Guangzhou Genio Biotechnology Co., Ltd.

[0028] Fetal bovine serum was purchased from Ecosai Biotechnology (Taicang) Co., Ltd.

[0029] High-glucose DMEM medium was purchased from Gibco.

[0030] The PDCoV-TJ1 strain was isolated and preserved by the Animal Disease Prevention and Control Innovation Team of the Institute of Animal Husbandry and Veterinary Medicine, Tianjin Academy of Agricultural Sciences; (Zheng Li, Li Xiuli, Yan Minghua*, Ren Weike, Zhang Lei, Lu Chao, Tian Xiangxue, Han Wei. Isolation, identification and biological characteristics analysis of porcine deltacoronavirus TJ1 strain [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine (Chinese core journal). 2018, 45(1):219-224);

[0031] The riboSCRIPT Reverse Transcription Kit (500T) was purchased from Guangzhou Ribobio Biotechnology Co., Ltd.

[0032] The Bulge-Loop™ miRNA qRT-PCR Starter Kit was purchased from Guangzhou Ribo Biotechnology Co., Ltd.

[0033] Opti-MEM medium was purchased from Thermo Fisher Scientific.

[0034] Lipofectamine 3000 Reagent was purchased from Thermo Fisher Scientific.

[0035] PrimeScript TM The RT reagent Kit (Perfect Real Time) was purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0036] TB Premix Ex Taq TM II (Tli RNaseH Plus) was purchased from Baori Biotechnology (Beijing) Co., Ltd.;

[0037] The laboratory reagents and supplies not mentioned are standard laboratory reagents and supplies, purchased from commercial channels;

[0038] Experimental methods not mentioned are standard experimental methods and will not be described in detail here.

[0039] Example 1

[0040] PDCoV infection of IPEC-J2 cells resulted in significant viral proliferation.

[0041] 1. Experimental cell culture:

[0042] IPEC-J2 cells were seeded in 6-well plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until a cell monolayer with 80% confluence was formed.

[0043] 2. Virus inoculation:

[0044] The PDCoV-TJ1 strain was inoculated into IPEC-J2 cells at an MOI of 1.0 (1.6 mL per well, containing 1% secretin in a final volume) (inoculation group), while a control group without virus inoculation was set up. The cells were then cultured at 37°C and 5% CO2.

[0045] 3. Total RNA extraction and cDNA product synthesis:

[0046] Total RNA was extracted using the TRIzol method. PrimeScript TM The RT reagent kit (Perfect Real Time) reverse transcribes total RNA into cDNA. 1 μg of total RNA is added to every 20 μL of system. The reverse transcription program is 30℃ for 10 min, 42℃ for 1 h, and 99℃ for 5 min.

[0047] 4. Quantitative Real-Time PCR:

[0048] The cDNA product obtained by reverse transcription was used as a template for quantitative real-time PCR; the PDCoV M gene primers and probes were diluted to 10 μM, respectively. The nucleotide sequences of the upstream primer, downstream primer, and probe of the PDCoV M gene are SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. Follow the instructions in the Probe qPCR Master Mix reagent manual to prepare the system and set the program (annealing temperature set to 61℃). Use this kit to complete the real-time PCR experiment.

[0049] Based on this standard curve equation, the Ct value obtained from the real-time PCR experiment is substituted into the real-time PCR linear equation: Ct value = -3.68 × lgX + 41.21, and the initial copy number of the sample can be calculated to achieve the purpose of quantification.

[0050] CGTGTGATCTATGTTATTAAAC, SEQ ID NO.3;

[0051] CAGGATATGAAGGTCAGTA, SEQ ID NO.4;

[0052] FAM-CTGCTCCAACCCTTCACCCTA-TAMRA, SEQ ID NO. 5.

[0053] The results are as follows Figure 1 As shown, Figure 1 The results showed that the expression level of the PDCoV M gene was increased in IPEC-J2 cells 24 h after PDCoV TJ1 infection.

[0054] Example 2

[0055] PDCoV infection of IPEC-J2 cells reduced the expression of ssc-miR-148a-5p.

[0056] 1. Experimental cell culture:

[0057] IPEC-J2 cells were seeded in 6-well plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until a cell monolayer with 80% confluence was formed.

[0058] 2. Virus inoculation:

[0059] The PDCoV-TJ1 strain was inoculated into IPEC-J2 cells at an MOI of 1.0 (1.6 mL per well, containing 1% secretin in a final volume) (inoculation group), while a control group without virus inoculation was set up. The cells were then cultured at 37°C and 5% CO2.

[0060] 3. Total RNA extraction and miRNA RT product synthesis:

[0061] IPEC-J2 cell pellets were collected 24 h after viral inoculation. Total RNA was extracted using the TRIzol method and reverse transcribed into miRNA RT products using the riboSCRIPT Reverse Transcription Kit (500T). The nucleotide sequence of the ssc-miR-148a-5p stem-loop primer used for reverse transcription was SEQ ID NO.6. 5 μg of total RNA was added to every 10 μL of the system. The reverse transcription program was 42℃ for 60 min and 70℃ for 10 min.

[0062] GTTGGGAGGTAGGAGGTTGATATCCTCCCAACAGTCGG, SEQ ID NO. 6.

[0063] 4. ssc-miR-148a-5p real-time PCR:

[0064] The miRNA RT product obtained by reverse transcription was used as a template for quantitative real-time PCR. Primers for ssc-miR-148a-5p and the internal reference gene U6 were diluted to 200 nM. The nucleotide sequences of the upstream and downstream primers for ssc-miR-148a-5p are SEQ ID NO.7 and SEQ ID NO.8, respectively, and the nucleotide sequences of the upstream and downstream primers for the internal reference gene U6 are SEQ ID NO.9 and SEQ ID NO.10, respectively. The Bulge-Loop™ miRNA qRT-PCR Starter Kit was used, and the system and program were configured according to the instructions (annealing temperature set to 60℃). Based on the Ct value of the quantitative real-time PCR experiment, U6 was used as the internal reference gene, and 2... -ΔΔCt The formula calculates the fold change in ssc-miR-148a-5p expression between the control group and the virus-treated group.

[0065] AGGCAAAGTTCTGAGACACT, SEQ ID NO.7;

[0066] TTGGGAGGTAGGAGGTTGAT, SEQ ID NO.8;

[0067] ATAGATCTAGGAGGACTCCAGGGAC, SEQ ID NO.9;

[0068] CTGAATTCGGGTCTTCTCAGAGG, SEQ ID NO. 10.

[0069] The results are as follows Figure 2 As shown, Figure 2The results showed that the expression level of ssc-miR-148a-5p was significantly reduced in IPEC-J2 cells 24 h after PDCoV TJ1 infection.

[0070] Example 3

[0071] Increased expression of ssc-miR-148a-5p inhibited the proliferation of PDCoV in IPEC-J2 cells.

[0072] 1. Experimental cell culture:

[0073] IPEC-J2 cells were seeded in 96-well plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until a cell monolayer with 80% confluence was formed.

[0074] 2. Cell transfection and infection experiments:

[0075] 0.75 μL of a 150 nM mimic (nucleotide sequence as shown in SEQ ID NO.1) and a mimic control were added to 5 μL of Opti-MEM medium and mixed thoroughly, then incubated for 10 min. 0.3 μL of Lipofectamine 3000 Reagent was added to 5 μL of Opti-MEM medium and mixed thoroughly, then incubated for 10 min. The diluted mimic solution and mimic control solution were then mixed thoroughly with the Lipofectamine 3000 Reagent added to Opti-MEM medium and incubated for 15 min. IPEC-J2 cells pre-seeded in 96-well plates were washed with Opti-MEM medium. The mimic / control and Lipofectamine were then added to the mixture. Add 90 μL of the 3000 Reagent mixture to each well of the cells, gently shake, and incubate in a cell culture incubator at 37°C and 5% CO2. After 24 h, inoculate the PDCoV-TJ1 strain into IPEC-J2 cells at an MOI of 1.0 (100 μL per well, containing 1% secretin at a final volume concentration). Then, incubate at 37°C and 5% CO2. Harvest the IPEC-J2 cell pellet 24 h after inoculation.

[0076] AAAGUUCUGAGACACUCCGACU, SEQ ID NO.1.

[0077] 3. Total RNA extraction, miRNA RT product and cDNA product synthesis:

[0078] Total RNA was extracted using the TRIzol method. The total RNA was reverse transcribed into miRNA RT products using the riboSCRIPT Reverse Transcription Kit (500T). The nucleotide sequence of the ssc-miR-148a-5p stem-loop primer used for reverse transcription was SEQ ID NO. 6. 5 μg of total RNA was added to every 10 μL of the system. The reverse transcription program was 42℃ for 60 min, followed by 70℃ for 10 min. (PrimeScript) TM The RT reagent kit (Perfect Real Time) reverse transcribes total RNA into cDNA. 1 μg of total RNA is added to every 20 μL of system. The reverse transcription program is 30℃ for 10 min, 42℃ for 1 h, and 99℃ for 5 min.

[0079] 4. ssc-miR-148a-5p real-time PCR:

[0080] The miRNA RT product obtained by reverse transcription was used as a template for quantitative real-time PCR. Primers for ssc-miR-148a-5p and the internal reference gene U6 were diluted to 200 nM. The nucleotide sequences of the upstream and downstream primers for ssc-miR-148a-5p are SEQ ID NO.7 and SEQ ID NO.8, respectively, and the nucleotide sequences of the upstream and downstream primers for the internal reference gene U6 are SEQ ID NO.9 and SEQ ID NO.10, respectively. The Bulge-Loop™ miRNA qRT-PCR Starter Kit was used, and the system and program were configured according to the instructions (annealing temperature set to 60℃). Based on the Ct value of the quantitative real-time PCR experiment, U6 was used as the internal reference gene, and 2... -ΔΔCt The formula calculates the fold change in ssc-miR-148a-5p expression between the simulant group and the simulant control group.

[0081] 5. PDCoV M gene real-time quantitative PCR:

[0082] The cDNA product obtained by reverse transcription was used as a template for quantitative real-time PCR. Primers for the PDCoV M gene and the internal reference gene GAPDH were diluted to 10 μM. The nucleotide sequences of the upstream and downstream primers for the PDCoV M gene were SEQ ID NO.3 and SEQ ID NO.4, respectively, and the nucleotide sequences of the upstream and downstream primers for the internal reference gene GAPDH were SEQ ID NO.11 and SEQ ID NO.12, respectively. (Based on TB...) Premix Ex Taq TMFollowing the instructions for the II (Tli RNaseH Plus) kit, configure the system and set the procedure (annealing temperature set to 59℃). Use this kit to perform real-time PCR experiments. Based on the Ct value of the real-time PCR experiment, use GAPDH as the internal reference gene and 2... -ΔΔCt The formula calculates the fold change in PDCoVM gene expression between the simulant group and the simulant control group.

[0083] AGGAGTAAGAGCCCCTGGA, SEQ ID NO.11;

[0084] TCTGGGATGGAAACTGGAA, SEQ ID NO. 12.

[0085] The results are as follows Figure 3 and Figure 4 As shown, Figure 3 The results showed that 24 h after transfection with a mimic that increased ssc-miR-148a-5p expression, the expression level of ssc-miR-148a-5p in PDCoV-infected IPEC-J2 cells was significantly increased. Figure 4 The results showed that in IPEC-J2 cells with increased ssc-miR-148a-5p expression, the expression level of the PDCoV M gene was significantly decreased, indicating a significant reduction in PDCoV proliferation. This suggests that increasing ssc-miR-148a-5p expression inhibits PDCoV proliferation in IPEC-J2 cells.

[0086] Example 4

[0087] Decreased expression of ssc-miR-148a-5p promotes the proliferation of PDCoV in IPEC-J2 cells.

[0088] 1. Experimental cell culture:

[0089] IPEC-J2 cells were seeded in 96-well plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until a cell monolayer with 80% confluence was formed.

[0090] 2. Cell transfection and infection experiments:

[0091] 0.75 μL of an inhibitor (nucleotide sequence shown in SEQ ID NO.2) that reduces ssc-miR-148a-5p expression to a final concentration of 150 nM and an inhibitor control were added to 5 μL of Opti-MEM medium and mixed thoroughly, then incubated for 10 min. 0.3 μL of Lipofectamine 3000 Reagent was added to 5 μL of Opti-MEM medium and mixed thoroughly, then incubated for 10 min. The diluted inhibitor solution and inhibitor control solution were then mixed thoroughly with the Lipofectamine 3000 Reagent added to Opti-MEM medium and incubated for 15 min. IPEC-J2 cells pre-seeded in 96-well plates were washed with Opti-MEM medium. Then, the above inhibitor / inhibitor control and Lipofectamine were added to the medium. Add 90 μL of the 3000 Reagent mixture to each well of the cells, gently shake, and incubate in a cell culture incubator at 37°C and 5% CO2. After 24 h, inoculate the PDCoV-TJ1 strain into IPEC-J2 cells at an MOI of 1.0 (100 μL per well, containing 1% secretin). Then, incubate at 37°C and 5% CO2. Harvest the IPEC-J2 cell pellet 24 h after inoculation.

[0092] AGTCGGAGTGTCTCAGAACTTT, SEQ ID NO. 2.

[0093] 3. Total RNA extraction, miRNA RT product and cDNA product synthesis:

[0094] Total RNA was extracted using the TRIzol method. The total RNA was reverse transcribed into miRNA RT products using the riboSCRIPT Reverse Transcription Kit (500T). The nucleotide sequence of the ssc-miR-148a-5p stem-loop primer used for reverse transcription was SEQ ID NO. 6. 5 μg of total RNA was added to every 10 μL of the system. The reverse transcription program was 42℃ for 60 min, followed by 70℃ for 10 min. (PrimeScript) TM The RT reagent kit (Perfect Real Time) reverse transcribes total RNA into cDNA. 1 μg of total RNA is added to every 20 μL of system. The reverse transcription program is 30℃ for 10 min, 42℃ for 1 h, and 99℃ for 5 min.

[0095] 4. ssc-miR-148a-5p real-time PCR:

[0096] The miRNA RT product obtained by reverse transcription was used as a template for quantitative real-time PCR. Primers for ssc-miR-148a-5p and the internal reference gene U6 were diluted to 200 nM. The nucleotide sequences of the upstream and downstream primers for ssc-miR-148a-5p are SEQ ID NO.7 and SEQ ID NO.8, respectively, and the nucleotide sequences of the upstream and downstream primers for the internal reference gene U6 are SEQ ID NO.9 and SEQ ID NO.10, respectively. The Bulge-Loop™ miRNA qRT-PCR Starter Kit was used, and the system and program were configured according to the instructions (annealing temperature set to 60℃). Based on the Ct value of the quantitative real-time PCR experiment, U6 was used as the internal reference gene, and 2... -ΔΔCt The formula calculates the fold change in ssc-miR-148a-5p expression between the inhibitor group and the inhibitor control group.

[0097] 5. PDCoV M gene real-time quantitative PCR:

[0098] The cDNA product obtained by reverse transcription was used as a template for quantitative real-time PCR. Primers for the PDCoV M gene and the internal reference gene GAPDH were diluted to 10 μM. The nucleotide sequences of the upstream and downstream primers for the PDCoV M gene were SEQ ID NO.3 and SEQ ID NO.4, respectively, and the nucleotide sequences of the upstream and downstream primers for the internal reference gene GAPDH were SEQ ID NO.11 and SEQ ID NO.12, respectively. (Based on TB...) Premix Ex Taq TM Following the instructions for the II (Tli RNaseH Plus) kit, configure the system and set the procedure (annealing temperature set to 59℃). Use this kit to perform real-time PCR experiments. Based on the Ct value of the real-time PCR experiment, use GAPDH as the internal reference gene and 2... -ΔΔCt The formula calculates the fold change in PDCoVM gene expression between the inhibitor group and the inhibitor control group.

[0099] The results are as follows Figure 5 and Figure 6 As shown, Figure 5 The results showed that 24 hours after transfection with an inhibitor that reduced ssc-miR-148a-5p expression, the expression level of ssc-miR-148a-5p in PDCoV-infected IPEC-J2 cells was significantly reduced. Figure 6The results showed that in IPEC-J2 cells with suppressed ssc-miR-148a-5p expression, the expression level of the PDCoV M gene was significantly increased, indicating a significant increase in PDCoV proliferation. This suggests that inhibiting ssc-miR-148a-5p expression promotes PDCoV proliferation in IPEC-J2 cells.

[0100] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0101] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of ssc-miR-148a-5p in the preparation of anti-PDCoV proliferation drugs; The nucleotide sequence of the ssc-miR-148a-5p is shown in SEQ ID NO.1; AAAGUUCUGAGACACUCCGACU, SEQ ID NO.1; The PDCoV in question is the PDCoV-TJ1 strain.

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