Use of overexpression agent of trims gene in preparation of product for inhibiting replication of porcine senecavirus

Abstract

The application discloses application of a TRIM gene overexpression agent in preparation of a product for inhibiting porcine Senecavirus replication. The application finds that after promoting the expression amount of the TRIM gene to be up-regulated, the virus replication capability in cells infected with the porcine Senecavirus is significantly inhibited; overexpression of the TRIM gene can cause significant decrease of gene expression and virus titer of the porcine Senecavirus. Based on this, an siRNA capable of inhibiting expression of the TRIM5 gene is designed, a nucleotide sequence of the siRNA is shown as SEQ ID NO:1; the siRNA can inhibit up-regulation of the expression amount of the TRIM5 gene in cells, and further promote replication of the porcine Senecavirus, thereby providing a high-titer virus source for research on a porcine Senecavirus vaccine.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, in particular to application of a TRIM gene overexpression agent in preparing a product for inhibiting porcine Seneca virus replication. Background Art

[0002] Senecavirus A (SVA) is a single-stranded, positive-sense RNA virus, the only member of the genus Senecavirus in the family Picornaviridae. Its genome is approximately 7.3 kb long and shares many structural similarities with other picornaviruses. The complete SVA virion is a naked, symmetrical icosahedral structure, 25-30 nm in diameter, and spherical in appearance. As a newly emerging infectious virus, SVA can cause idiopathic vesicular disease in pigs, severely impacting the economic profitability of pig farms and posing a significant threat to the safety of the pig farming industry.

[0003] Research Progress on Porcine Seneca Virus: The viral genome contains only one open reading frame (ORF), with a type IV internal ribosome entry site in the 5' noncoding region mediating translation of the virus's single ORF into a polyprotein. However, limited research exists on the pathogenicity of porcine Seneca Virus, particularly on targeted genes that interact with the virus. This has led to a lack of effective viral prevention and control strategies. Therefore, identifying targeted genes that interact with the virus is crucial for understanding its pathogenesis.

[0004] The tripartite motif protein (TRIM) family is present in almost all multicellular animals and can participate in the collective innate immune response as immunomodulatory proteins and E3 ubiquitin ligases. With the continuous deepening of research, it has been found that the TRIM protein family not only maintains the body's normal physiological functions, such as cell growth, membrane repair, signaling pathways, and apoptosis, but also participates in the regulation of various diseases. In addition, TRIM5 protein has excellent anti-HIV ability, which is currently the most extensive TRIM5 antiviral research. However, HIV and porcine Seneca virus belong to two completely different types of viruses, and there are currently no studies related to TRIM anti-porcine Seneca virus. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide an application of a TRIM gene overexpression agent in the preparation of a product for inhibiting the replication of porcine Seneca virus.

[0006] The first object of the present invention is to provide use of an agent for promoting TRIM gene expression in the preparation of a product against porcine Seneca virus.

[0007] The second object of the present invention is to provide use of an agent for promoting TRIM protein expression in the preparation of a product against porcine Seneca virus.

[0008] The third object of the present invention is to provide use of an agent for promoting the expression of TRIM protein and / or TRIM gene in the preparation of a drug for treating and / or resisting porcine Seneca virus infection.

[0009] The fourth object of the present invention is to provide the use of siRNA having a nucleotide sequence as shown in SEQ ID NO: 1 in the preparation of porcine Seneca virus vaccine.

[0010] The fifth object of the present invention is to provide use of a recombinant vector containing siRNA having a nucleotide sequence as shown in SEQ ID NO: 1 in the preparation of porcine Seneca virus.

[0011] The sixth object of the present invention is to provide a drug for resisting porcine Seneca virus.

[0012] In order to achieve the above object, the present invention is implemented through the following scheme:

[0013] Application of a reagent for promoting TRIM gene expression in the preparation of a product against porcine Seneca virus.

[0014] Preferably, the TRIM gene is the TRIM5 gene.

[0015] Preferably, the anti-Senecavirus product is a drug that inhibits the replication of Senecavirus.

[0016] Application of a reagent for promoting TRIM protein expression in the preparation of a product against porcine Seneca virus.

[0017] Preferably, the TRIM protein is TRIM5 protein.

[0018] The present invention also claims the use of an agent for promoting the expression of TRIM protein and / or TRIM gene in the preparation of a drug for treating and / or resisting porcine Seneca virus infection.

[0019] The inventors used metagenomics to study the relationship between the TRIM5 gene, a member of the TRIM protein family, and porcine Senecavirus replication in a cell. They unexpectedly discovered that inhibiting TRIM5 gene expression significantly promoted porcine Senecavirus replication, while promoting or upregulating TRIM5 gene expression significantly inhibited porcine Senecavirus replication. Based on this, the inventors designed an siRNA capable of inhibiting TRIM5 gene expression. The nucleotide sequence of the siRNA is shown in SEQ ID NO: 1. The siRNA specifically binds to the TRIM5 gene and forms a hairpin structure, thereby interfering with TRIM5 gene transcription and reducing TRIM5 protein expression.

[0020] A siRNA for inhibiting up-regulation of TRIM5 gene expression, wherein the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1.

[0021] siRNA (SEQ ID NO: 1): GGACGAGGAGAAAGTTATTC.

[0022] A recombinant vector for inhibiting TRIM5 gene expression, wherein the recombinant vector is recombined with the above-mentioned siRNA.

[0023] The present invention also claims to protect the use of siRNA with a nucleotide sequence as shown in SEQ ID NO: 1 in the preparation of porcine Seneca virus vaccine.

[0024] The present invention also claims the use of a recombinant vector containing siRNA having a nucleotide sequence as shown in SEQ ID NO: 1 in the preparation of porcine Seneca virus.

[0025] The siRNA with a nucleotide sequence as shown in SEQ ID NO: 1 and / or a recombinant vector containing the siRNA with a nucleotide sequence as shown in SEQ ID NO: 1 can inhibit the expression of the TRIM5 gene, thereby enhancing the replication ability of porcine Seneca virus, providing a stable virus source and high-titer virus for the vaccine development process.

[0026] The present invention also claims protection for a drug against porcine Seneca virus, which inhibits the replication of porcine Seneca virus by promoting the expression of TRIM gene and / or TRIM protein.

[0027] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention provides the use of an overexpression agent of the TRIM gene in the preparation of a product for inhibiting the replication of porcine Seneca virus. The present invention has found through research that after promoting the upregulation of the expression of the TRIM gene, the viral replication ability in cells infected with porcine Seneca virus is significantly inhibited; overexpression of the TRIM gene leads to a significant decrease in the gene expression and viral titer of porcine Seneca virus. Based on this, a siRNA capable of inhibiting the expression of the TRIM5 gene is designed, the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1; the siRNA can inhibit the upregulation of the expression of the TRIM5 gene in cells, thereby promoting the replication of porcine Seneca virus, and providing a high-titer virus source for the study of porcine Seneca virus vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the growth curve of the SVA virus in Example 1;

[0031] Figure 2 This is a diagram of the fluorescence quantitative detection results in Example 1;

[0032] Figure 3 This is a diagram showing the results of double enzyme digestion electrophoresis identification of the pEGFP-N1-TRIM5 plasmid in Example 2;

[0033] Figure 4 Figure 2 is the observation and detection diagram of 3D4 / 21 cells transfected with pEGFP-N1-TRIM5 plasmid; a is the fluorescence microscopy observation diagram of 3D4 / 21 cells transfected with pEGFP-N1 plasmid; b is the fluorescence microscopy observation diagram of 3D4 / 21 cells transfected with pEGFP-N1-TRIM5 plasmid; c is the detection result of qRT-PCR; d is the detection result of Western Blot;

[0034] Figure 5 Figure 2 is the SVA replication status of 3D4 / 21 cells transfected with pEGFP-N1-TRIM5 plasmid and 3D4 / 21 cells transfected with pEGFP-N1 plasmid after infection with porcine Seneca virus; a is the result of qRT-PCR; b is TCID 50 Method detection results; c is the Western Blot detection result;

[0035] Figure 6 This is a diagram showing the double enzyme digestion electrophoresis identification results of the sh-TRIM5 plasmid in Example 3;

[0036] Figure 7Figure 2 is the observation and detection image of 3D4 / 21 cells transfected with sh-TRIM5 plasmid; a is the fluorescence microscopy observation image of 3D4 / 21 cells transfected with sh-NC plasmid; b is the fluorescence microscopy observation image of 3D4 / 21 cells transfected with sh-TRIM5 plasmid; c is the detection result of qRT-PCR; d is the detection result of Western Blot;

[0037] Figure 8 Figure 3 is the SVA replication of 3D4 / 21 cells transfected with sh-TRIM5 plasmid and 3D4 / 21 cells transfected with sh-NC plasmid after infection with porcine Seneca virus; a is the detection result of qRT-PCR; b is TCID 50 Method detection results; c is the Western Blot detection result. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0039] Example 1 Screening of porcine Seneca virus replication-related genes

[0040] 1. Experimental methods

[0041] (1) Determination of the growth curve of SVA-infected cells

[0042] The revived PK-15 cells were inoculated into DMEM medium containing 10% FBS (v / v) and cultured at 37°C, 5% CO2 for 24 h to obtain cultured 3D4 / 21 cells. The cultured 3D4 / 21 cells were plated at 5.0×10 5 The cells were inoculated into a multi-well culture dish at a density of 100 cells / well. The next day, after the cells adhered to the wall, the cells were inoculated with porcine Seneca virus (GH-GDFS-2018) at an inoculation dose of MOI = 1.0, placed in a 37°C cell culture incubator for 2 hours, and then the medium was changed. Then, the cells were placed in a 37°C cell culture incubator for further culture. The cells and supernatant were collected after 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours and 72 hours of culture, respectively. The samples at each time point were repeatedly frozen and thawed 3 times, and then the samples were diluted 10 times and inoculated into cells. Then, the tissue half-infectious lethal dose (TCID 50 ) method to determine the corresponding virus titer, and the corresponding SVA virus growth curve was drawn using GraphPad Prism7 software.

[0043] (2) mRNA metagenomic sequencing of cells infected with SVA

[0044] According to the SVA virus growth curve obtained in step (1), the different time points of SVA virus infection of cells were determined and divided into a blank control group (not infected with the virus), an early slow proliferation group (0 to 12 h), a mid-stage rapid proliferation group (12 to 6 h), and a late slow proliferation group (36 to 72 h).

[0045] Virus-infected cells from different groups were collected and RNA was extracted separately. After the extraction, metagenomic sequencing of mRNA was performed. The raw data obtained by metagenomic sequencing were subjected to sequencing quality control and sequencing base quality value determination. Then, the sequencing data of virus-infected cells in different groups were analyzed for mRNA differential expression, and the functions of the differentially expressed genes were annotated and enriched.

[0046] (3) Fluorescence quantitative verification

[0047] According to the TRIM5 gene sequence on NCBI (NM_001044532.1), the fluorescence quantitative primers shown in Table 1 were designed.

[0048] Table 1 Fluorescence quantitative primers

[0049] The cultured PK-15 cells obtained in step (1) were infected with porcine Seneca virus at an inoculation dose of MOI = 1.0, and cell samples were collected at 0 h, 6 h, 12 h, 24 h, 36 h and 48 h after infection, and total RNA was extracted. The corresponding cDNA was obtained by reverse transcription as a template, and qRT-PCR amplification was performed using TRIM5-F with a nucleotide sequence as shown in SEQ ID NO: 4 and TRIM5-R with a nucleotide sequence as shown in SEQ ID NO: 5 in Table 1 to detect changes in TRIM5 gene expression; GAPDH was detected as an internal reference using GAPDH-F with a nucleotide sequence as shown in SEQ ID NO: 2 and GAPDH-R with a nucleotide sequence as shown in SEQ ID NO: 3.

[0050] The amplification system for qRT-PCR amplification is: 10 μL Green Pro Taq HS Premix, 0.5 μL of TRIM5-F (SEQ ID NO: 4), 0.5 μL of TRIM5-R (SEQ ID NO: 5), 4 μL of template, and RNase-free dH2O were added to a total volume of 20 μL.

[0051] The amplification program for qRT-PCR amplification was: 95°C, 30 s; 95°C, 5 s, 60°C, 30 s, 40 cycles.

[0052] Cultured 3D4 / 21 cells not infected with SVA virus were used as a blank control group (Control), and cultured 3D4 / 21 cells infected with dead SVA virus were used as a negative control group (Heat-SVA), and the changes in TRIM5 gene expression were detected in the same manner.

[0053] 2. Experimental results

[0054] The growth curve of SVA virus is shown in the figure Figure 1 As shown, the results showed that the virus multiplied and amplified rapidly 3 to 36 hours after SVA infected 3D4 / 21 cells, and reached the peak of virus titer 36 hours after infection, and then the virus titer began to slowly decline.

[0055] Fluorescence quantitative detection results Figure 2 As shown, the results showed that the gene expression of TRIM5 in 3D4 / 21 cells after SVA infection increased significantly and reached a peak at 24 hours.

[0056] Example 2 Effect of TRIM5 gene expression on porcine Seneca virus

[0057] 1. Experimental methods

[0058] The TRIM5 gene sequence (NM_001044532.1) on NCBI was synthesized and ligated into the EcoRI restriction site of the pMD18T vector to obtain the pMD18T-TRIM5 recombinant plasmid.

[0059] Using the pMD18T-TRIM5 recombinant plasmid as a template, PCR amplification was performed using the nucleotide sequence of TRIM5-CDS-F shown in SEQ ID NO: 8 and the nucleotide sequence of TRIM5-CDS-R shown in SEQ ID NO: 9. The PCR amplification products were detected by gel electrophoresis. The electrophoretic band of the PCR amplification product was approximately 1500 bp, which was close to the size of the TRIM5 gene fragment itself. The PCR amplification product was the TRIM5 gene fragment containing the restriction enzyme cleavage site.

[0060] The PCR amplification system was as follows: 5 μL of 2×Accurate Taq, 0.5 μL of TRIM5-CDS-F (SEQ ID NO: 8), 0.5 μL of TRIM5-CDS-R (SEQ ID NO: 9), 2 μL of template, and 2 μL of sterile double-distilled water.

[0061] The PCR amplification program was as follows: 95°C, 5 min; 95°C, 30 s, 65°C, 30 s, 72°C, 100 s, 38 cycles; 72°C, 5 min; and hold at 4°C.

[0062] TRIM5-CDS-F (SEQ ID NO: 8):

[0063] 5'-CCG CTCGAGG CCACCATGGCTTCAGGCATCCTGGA-3';

[0064] TRIM5-CDS-R (SEQ ID NO: 9):

[0065] 5'-CCG GAATTCG AGAGCCCGGCGAGCACAGA-3'.

[0066] The PCR amplification product was recovered and double-enzyme digested with the pEGFP-N1 vector using XhoI endonuclease and EcoRI endonuclease, respectively, to obtain the digested PCR amplification product and the linearized pEGFP-N1 vector. Subsequently, T4 DNA ligase was used to ligate the product to obtain the ligation product, which was transformed into E. coli JM109 competent cells and cultured in solid LB medium at 37°C. After inverted culture for 12 hours, a single clone colony was picked for strain proliferation. The plasmid was then extracted using an endotoxin-free plasmid extraction kit and double-enzyme digested with XhoI endonuclease and EcoRI endonuclease for identification. The correct plasmid was identified as the pEGFP-N1-TRIM5 plasmid.

[0067] The pEGFP-N1-TRIM5 plasmid was transfected into 3D4 / 21 cells and observed under a fluorescence microscope. The expression of TRIM5 gene in the transfected cells was detected by qRT-PCR and Western Blot using TRIM5-F (SEQ ID NO: 4) and TRIM5-R (SEQ ID NO: 5), respectively.

[0068] 24 h after transfection, 3D4 / 21 cells transfected with pEGFP-N1-TRIM5 plasmid were infected with porcine Seneca virus at an inoculation dose of MOI = 1.0. Cell samples were collected at 6 h, 12 h, 24 h, 36 h, and 48 h after infection, and RNA and protein in the cell samples were extracted. qRT-PCR and TCID were performed using SVA-F (SEQ ID NO: 6) and SVA-R (SEQ ID NO: 7), respectively. 50 The replication of SVA in cell samples was detected by Western Blot.

[0069] The control group was set up as follows: pEGFP-N1 plasmid was transfected into 3D4 / 21 cells, and the cells were treated in the same way and the expression of TRIM5 and the replication of SVA in the cells were detected.

[0070] 2. Experimental results

[0071] The results of enzyme digestion identification are as follows Figure 3 As shown, the results showed that the sizes of the two electrophoresis bands were approximately 4700 bp and 1489 bp, respectively, which was consistent with the expected results, indicating that the recombinant plasmid was successfully constructed.

[0072] The observation and detection diagram of pEGFP-N1-TRIM5 plasmid transfected into 3D4 / 21 cells is shown in the figure Figure 4 As shown, a is a fluorescence microscopy observation of 3D4 / 21 cells transfected with pEGFP-N1 plasmid; b is a fluorescence microscopy observation of 3D4 / 21 cells transfected with pEGFP-N1-TRIM5 plasmid; c is the detection result of qRT-PCR, where vector is the detection result of pEGFP-N1 vector; d is the detection result of Western Blot.

[0073] The results showed that the plasmid was successfully transfected into 3D4 / 21 cells, and the expression level of TRIM5 was significantly increased in the transfected cells.

[0074] The SVA replication of 3D4 / 21 cells transfected with pEGFP-N1-TRIM5 plasmid and 3D4 / 21 cells transfected with pEGFP-N1 plasmid after infection with porcine Seneca virus is shown in Figure 2. Figure 5 As shown, a is the detection result of qRT-PCR; b is TCID 50 Method detection results; c is the Western Blot detection result.

[0075] The results showed that compared with the control group, the gene copy number and viral titer of SVA were significantly decreased after cells transfected with pEGFP-N1-TRIM5 plasmid overexpressed TRIM5, indicating that promoting the upregulation of TRIM5 expression would significantly inhibit the replication of porcine Seneca virus.

[0076] Example 3: An siRNA that improves the replication ability of porcine Seneca virus

[0077] 1. Experimental methods

[0078] Using the TRIM5 gene sequence (NM_001044532.1) on NCBI as the target gene, shTRIM5-F as shown in SEQ ID NO: 10 and shTRIM5-R as shown in SEQ ID NO: 11 were designed, and siRNA as shown in SEQ ID NO: 1 was synthesized by oligo annealing.

[0079] shTRIM5-F (SEQ ID NO: 10):

[0080] 5'-GATCCCCGGACGAGGAGAAAGTTATTCTttcaagagaAGAATAACTTTCTCCTCGT CCTTTTTA-3';

[0081] shTRIM5-R (SEQ ID NO: 11):

[0082] 5'-AGCTTAAAAAGGACGAGGAGAAAGTTATTCTtctcttgaaAGAATAACTTTCTCCT CGTCCGGG-3'.

[0083] siRNA (SEQ ID NO: 1): 5'-GGACGAGGAGAAAGTTATTC-3'.

[0084] The oligo annealing step is as follows: 1 μL each of shTRIM5-F represented by SEQ ID NO: 10 and shTRIM5-R represented by the nucleotide sequence of SEQ ID NO: 11 is added to 48 μL of annealing buffer, pipetted to mix, and annealed at 95°C for 5 minutes, 72°C for 15 minutes, and cooled naturally to room temperature to obtain siRNA (SEQ ID NO: 1).

[0085] The siRNA (SEQ ID NO: 1) and the interference vector (pSuper.Retro.Neo+GFP) were double-digested with Hind III endonuclease and EcoR I endonuclease, respectively, to obtain the digested siRNA and the linearized interference vector, which were then ligated using T4 DNA ligase to obtain a ligation product 1. The ligation product 1 was transformed into E. coli JM109 competent cells and cultured in LB medium at 37°C. Monoclonal colonies were picked and proliferated, the bacterial precipitates were collected, and the plasmids were extracted using an endotoxin removal kit. The cells were double-digested and identified by electrophoresis using Hind III endonuclease and EcoR I endonuclease.

[0086] The correct plasmid identified by electrophoresis was collected, namely the sh-TRIM5 plasmid. After sh-TRIM5 was transfected into 3D4 / 21 cells, the cells were observed under a fluorescence microscope. qRT-PCR and Western Blot were performed using TRIM5-F (SEQ ID NO: 4) and TRIM5-R (SEQ ID NO: 5), respectively, to detect the expression of the TRIM5 gene in the transfected cells.

[0087] 24 h after transfection, cells were infected with porcine Seneca virus (SVA) at an inoculation dose of MOI = 1.0. Cell samples were collected at different time points, including 6 h, 12 h, 24 h, 36 h, and 48 h after virus infection. RNA and protein in the cells were extracted, and qRT-PCR and TCID were performed using SVA-F (SEQ ID NO: 6) and SVA-R (SEQ ID NO: 7), respectively. 50 The replication of SVA in cell samples was detected by Western Blot.

[0088] The control group was set up as follows: 3D4 / 21 cells were transfected with an empty plasmid (ie, pSuper.Retro.Neo+GFP plasmid).

[0089] 2. Experimental results

[0090] The results of double enzyme electrophoresis identification were as follows Figure 6 As shown, the results showed that the sizes of the electrophoresis bands were approximately 7100 bp, 1130 bp and 281 bp, respectively, which met the requirements. The recombinant plasmid containing the siRNA shown in SEQ ID NO: 1 was successfully constructed.

[0091] The observation and detection diagram of sh-TRIM5 plasmid transfected into 3D4 / 21 cells is shown in the figure Figure 7 As shown, a is a fluorescence microscopy observation of 3D4 / 21 cells transfected with sh-NC plasmid; b is a fluorescence microscopy observation of 3D4 / 21 cells transfected with sh-TRIM5 plasmid; c is the detection result of qRT-PCR; d is the detection result of Western Blot.

[0092] The results showed that after the sh-TRIM5 plasmid was transfected into the cells, the expression level of TRIM5 in the cells was significantly reduced, indicating that the sh-TRIM5 plasmid can inhibit the expression of the TRIM5 gene.

[0093] The SVA replication of 3D4 / 21 cells transfected with sh-TRIM5 plasmid and 3D4 / 21 cells transfected with sh-NC plasmid after infection with porcine Seneca virus is shown in Figure 2. Figure 8 As shown; a is the detection result of qRT-PCR; b is TCID 50 Method detection results; c is the Western Blot detection result.

[0094] The results showed that after reducing the expression of TRIM5 in cells, the copy number and viral titer of SVA increased significantly, indicating that interfering with the expression of TRIM5 gene would promote the replication of SVA.

[0095] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of pMD18T-TRIM5 plasmid in the preparation of products against porcine Seneca virus; The pMD18T-TRIM5 plasmid was obtained by synthesizing the TRIM5 gene sequence from NCBI and ligating it into the EcoRI restriction site of the pMD18T vector; The accession number of the TRIM5 gene sequence in the NCBI database is NM_001044532.1.

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