CN117281824A - Application of overexpression agent of TRIM gene in preparation of product for inhibiting replication of porcine Session inner card virus - Google Patents
Application of overexpression agent of TRIM gene in preparation of product for inhibiting replication of porcine Session inner card virus Download PDFInfo
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Abstract
The invention discloses an application of an overexpression agent of TRIM genes in preparation of a product for inhibiting replication of porcine Session inner card virus. According to the invention, after the expression quantity of the TRIM gene is promoted to be up-regulated, the virus replication capacity in cells infected with the porcine Seika virus is obviously inhibited; overexpression of the TRIM gene results in a significant decrease in gene expression and viral titers of porcine saint virus. Based on the design, an siRNA capable of inhibiting TRIM5 gene expression is designed, and the nucleotide sequence of the siRNA is shown as SEQ ID NO:1 is shown in the specification; the siRNA can inhibit the up-regulation of the expression level of TRIM5 genes in cells, so that the replication of the porcine Seika virus is promoted, and a high-titer virus source is provided for researching the porcine Seika virus vaccine.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to application of an overexpression agent of TRIM genes in preparation of a product for inhibiting replication of porcine Senecard virus.
Background
Porcine Senecavirus A (SVA) is a single-stranded positive strand RNA virus belonging to the genus Senecavirus of the family Picornaviridae, the genome of which is about 7.3kb in length and has many similarities to other picornaviruses, and the complete SVA virion is a naked symmetrical icosahedral structure with a diameter of 25-30 nm and a spherical appearance. As a new infectious virus, the porcine Sesinkavirus can cause idiopathic vesicular disease to pigs after infecting pig groups, so that the economic benefit of pig farms is seriously affected, and great threat is brought to the safety of the breeding industry.
The genome of the porcine sai virus developed virus contained only 1 Open Reading Frame (ORF), and an internal ribosome entry site of type IV was present in the 5' non-coding region, mediating translation of the virus-unique ORF into polyprotein. However, the research on the pathogenic mechanism of the porcine Seika virus is less at present, especially, the research on the targeting gene interacting with the virus is less, so that a better virus prevention and control means is lacked, and therefore, the search of the targeting gene interacting with the virus has important significance for researching the pathogenic mechanism of the virus.
The triple motif protein family (Tripartite motif protein, TRIM) exists in almost all multicellular animals and is capable of participating in a collective innate immune response as an immunomodulatory protein and E3 ubiquitin ligase. With the continuous and intensive research, it is found that the TRIM protein family not only can maintain the normal physiological functions of organisms, such as cell growth, membrane repair, signal paths, apoptosis and the like, but also can participate in the regulation and control process of various diseases; in addition, TRIM5 protein has good anti-HIV virus capability, which is the most extensive TRIM5 antiviral study at present. However, the HIV virus and the Session card virus belong to two completely different viruses, and no research on the anti-Session card virus related to TRIM is currently seen.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the application of the overexpression agent of the TRIM gene in the preparation of products for inhibiting the replication of the porcine Session inner card virus.
The first object of the present invention is to provide the use of an agent promoting the expression of the TRIM gene for the preparation of a product against porcine sai virus.
A second object of the present invention is to provide the use of an agent promoting the expression of TRIM proteins for the preparation of a product against porcine sai virus.
A third object of the present invention is to provide the use of an agent promoting the expression of TRIM proteins and/or TRIM genes in the manufacture of a medicament for the treatment and/or resistance to infection by porcine sai virus.
A fourth object of the present invention is to provide a nucleic acid sequence as set forth in SEQ ID NO:1 in the preparation of a porcine sai virus vaccine.
A fifth object of the present invention is to provide a recombinant polypeptide having a nucleotide sequence as set forth in SEQ ID NO:1 in the preparation of porcine sai virus.
The sixth object of the invention is to provide a medicament for resisting porcine saikavirus.
In order to achieve the above object, the present invention is realized by the following means:
the application of an agent for promoting TRIM gene expression in preparing a product for resisting porcine saikovirus.
Preferably, the TRIM gene is a TRIM5 gene.
Preferably, the anti-swine saint virus product is a drug that inhibits replication of swine saint virus. .
Use of an agent that promotes expression of a TRIM protein in the preparation of a product against porcine saikovirus.
Preferably, the TRIM protein is TRIM5 protein.
The invention also claims the use of an agent that promotes the expression of a TRIM protein and/or TRIM gene in the manufacture of a medicament for the treatment and/or resistance to infection by porcine sai virus.
The inventors studied the relationship between the TRIM5 gene in TRIM protein family and replication of porcine sedum card virus in one cell using metagenome, and unexpectedly found that inhibiting the expression of TRIM5 gene significantly promoted replication of porcine sedum card virus, while promoting or up-regulating the expression of TRIM5 gene significantly inhibited replication of porcine sedum card virus. Based on this, the inventor designs an siRNA capable of inhibiting TRIM5 gene expression, and the nucleotide sequence of the siRNA is shown as SEQ ID NO:1 is shown in the specification; the siRNA can specifically bind with the TRIM5 gene and form a hairpin structure, so that transcription of the TRIM5 gene is interfered, and the expression quantity of the TRIM5 protein is reduced.
An siRNA for inhibiting up-regulation of TRIM5 gene expression, wherein the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1.
siRNA(SEQ ID NO：1)：GGACGAGGAGAAAGTTATTC。
A recombinant vector for inhibiting the expression of TRIM5 gene, wherein the recombinant vector is recombined with the siRNA.
The invention also claims a nucleotide sequence as set forth in SEQ ID NO:1 in the preparation of a porcine sai virus vaccine.
The invention also claims recombinant nucleotide sequences as shown in SEQ ID NO:1 in the preparation of porcine sai virus.
Wherein the nucleotide sequence is shown in SEQ ID NO:1 and/or the recombinant siRNA has a nucleotide sequence shown as SEQ ID NO:1 can inhibit TRIM5 gene expression, thereby improving replication capacity of the porcine Seika virus and providing stable virus source and high titer virus for vaccine research and development process.
The invention also claims a medicine for resisting the porcine Session inner card virus, which can inhibit the replication of the porcine Session inner card virus by promoting the expression of TRIM genes and/or TRIM proteins.
Preferably, the medicament further comprises a pharmaceutically acceptable carrier.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an application of an overexpression agent of TRIM genes in preparation of a product for inhibiting replication of porcine Session inner card virus. According to the invention, after the expression quantity of the TRIM gene is promoted to be up-regulated, the virus replication capacity in cells infected with the porcine Seika virus is obviously inhibited; overexpression of the TRIM gene results in a significant decrease in gene expression and viral titers of porcine saint virus. Based on the design, an siRNA capable of inhibiting TRIM5 gene expression is designed, and the nucleotide sequence of the siRNA is shown as SEQ ID NO:1 is shown in the specification; the siRNA can inhibit the up-regulation of the expression level of TRIM5 genes in cells, so that the replication of the porcine Seika virus is promoted, and a high-titer virus source is provided for researching the porcine Seika virus vaccine.
Drawings
FIG. 1 is a graph of SVA virus growth in example 1;
FIG. 2 is a graph showing the results of quantitative fluorescence detection in example 1;
FIG. 3 is a graph showing the results of double-enzyme digestion electrophoresis of pEGFP-N1-TRIM5 plasmid of example 2;
FIG. 4 is a diagram showing the observation of transfection of pEGFP-N1-TRIM5 plasmid into 3D4/21 cells; a is a fluorescence microscope view of 3D4/21 cells transfected with pEGFP-N1 plasmid; b is a fluorescence microscope view 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;
FIG. 5 is a graph showing 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 Seika virus; a is the detection result of qRT-PCR; b is TCID 50 Detecting a result by the method; c is a Western Blot detection result;
FIG. 6 is a graph showing the results of double enzyme digestion electrophoresis of the sh-TRIM5 plasmid of example 3;
FIG. 7 is a diagram showing the observation and detection of transfection of sh-TRIM5 plasmid into 3D4/21 cells; a is a fluorescence microscope observation diagram of 3D4/21 cells transfected with sh-NC plasmid; b is a fluorescence microscope observation diagram 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;
FIG. 8 is a graph showing SVA replication of 3D4/21 cells transfected with sh-TRIM5 plasmid and 3D4/21 cells transfected with sh-NC plasmid after infection with porcine Seika virus; a is the detection result of qRT-PCR; b is TCID 50 Detecting a result by the method; c is the Western Blot detection result.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 screening of porcine Session inner card Virus replication-associated Gene
1. Experimental method
(1) Determination of SVA infected cell growth curve
Resuscitated PK-15 cells were inoculated into DMEM medium containing 10% FBS (v/v) at 37℃with 5% CO 2 Culturing for 24 hr to obtain cultured 3D4/21 cells, and culturing 3D4/21 cells according to 5.0X10 5 Cell density of each well was inoculated into a multi-well dish, cells were inoculated with porcine Seikovia virus (GH-GDFS-2018) at an inoculum dose of MOI=1.0 after cell attachment every other day, incubated in a 37℃cell incubator for 2 hours, changed, then incubated in a 37℃cell incubator for continuous culture, cells and supernatant were collected after 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours and 72 hours, respectively, samples at each time point were repeatedly freeze-thawed 3 times, diluted by a 10-fold ratio and inoculated with cells, and then the cells were infected with half of the tissue to death (TCID 50 ) Corresponding viral titers were determined and corresponding SVA virus growth was plotted using GraphPad Prism7 software.
(2) mRNA metagenomic sequencing of cells after SVA infection
According to the SVA virus growth curve chart obtained in the step (1), different time points of SVA virus infected cells are determined and divided into a blank control group (uninfected virus), a early slow proliferation group (0-12 h), a middle fast proliferation group (12-6 h) and a later slow proliferation group (36-72 h).
Collecting virus-infected cells in different groups, respectively extracting RNA, performing metagenomic sequencing of mRNA after extraction, performing sequencing quality control and sequencing alkali matrix value measurement on the raw data obtained by metagenomic sequencing, performing mRNA differential expression analysis on the sequencing data in the virus-infected cells in different groups, and performing annotation and enrichment analysis on the functions of the obtained differential expression genes.
(3) Fluorescent quantitative verification
The fluorescent quantitative primers shown in Table 1 were designed based on the TRIM5 gene sequence (NM-001044532.1) at NCBI.
TABLE 1 fluorescent quantitative primers
Infecting the cultured PK-15 cells obtained in step (1) with the swine saint virus at an inoculum size of moi=1.0, collecting cell samples at 0h, 6h, 12h, 24h, 36h and 48h after infection, respectively, extracting total RNA, and reverse transcription to obtain the corresponding cDNA as a template, using the nucleotide sequences as set forth in SEQ ID NOs: 4 and the nucleotide sequence of TRIM5-F is shown as SEQ ID NO:5, performing qRT-PCR amplification on TRIM5-R, and detecting the change of the expression of the TRIM5 gene; the nucleotide sequence is shown as SEQ ID NO:2 and the nucleotide sequence of the GAPDH-F is shown as SEQ ID NO: GAPDH-R shown in FIG. 3 detects GAPDH as an internal reference.
Wherein the qRT-PCR amplification system comprises: 10 mu L ofGreen Pro Taq HS Premix, 0.5. Mu.L of TRIM5-F (SEQ ID NO: 4), 0.5. Mu.L of TRIM5-R (SEQ ID NO: 5), 4. Mu.L of template, RNase Free dH 2 O was made up to a total volume of 20. Mu.L.
The amplification procedure of qRT-PCR amplification is: 95 ℃ for 30s;95 ℃,5s,60 ℃,30s,40 cycles.
The change in TRIM5 gene expression was equally detected using post-culture 3D4/21 cells that had not been infected with SVA virus as a Control blank (Control) and post-culture 3D4/21 cells that had been infected with dead SVA virus as a negative Control (Heat-SVA).
2. Experimental results
The SVA virus growth graph is shown in FIG. 1, and the results show that: SVA rapidly proliferates and expands 3-36 hours after infection of 3D4/21 cells and reaches a peak viral titer period 36 hours after infection, after which the viral titer begins to slowly decline.
The fluorescence quantitative detection results are shown in fig. 2, and the results show that: in 3D4/21 cells after SVA infection, the gene expression level of TRIM5 increased significantly, peaking at 24 h.
Example 2 Effect of TRIM5 Gene expression on porcine Sesinkavirus
1. Experimental method
The TRIM5 gene sequence (NM-001044532.1) on NCBI was synthesized and ligated to the EcoRI cleavage site of the pMD18T vector to give the pMD18T-TRIM5 recombinant plasmid.
The pMD18T-TRIM5 recombinant plasmid is used as a template, and a nucleotide sequence shown as SEQ ID NO:8 and the nucleotide sequence of the TRIM5-CDS-F is shown as SEQ ID NO: and 9, performing PCR amplification on the TRIM5-CDS-R, and performing gel electrophoresis detection on a PCR amplification product, wherein the electrophoresis band of the PCR amplification product is about 1500bp and is close to the size of the TRIM5 gene fragment, and the PCR amplification product is the TRIM5 gene fragment containing the enzyme cutting site.
Wherein the PCR amplification system is as follows: mu.L of 2 XAcurate Taq, 0.5. Mu.L of TRIM5-CDS-F (SEQ ID NO: 8), 0.5. Mu.L of TRIM5-CDS-R (SEQ ID NO: 9), 2. Mu.L of template, 2. Mu.L of sterile double distilled water.
The PCR amplification procedure was: 95 ℃ for 5min;95 ℃,30s,65 ℃,30s,72 ℃,100s,38 cycles; 72 ℃ for 5min; maintained at 4 ℃.
TRIM5-CDS-F(SEQ ID NO：8)：
5’-CCGCTCGAGGCCACCATGGCTTCAGGCATCCTGGA-3’；
TRIM5-CDS-R(SEQ ID NO：9)：
5’-CCGGAATTCGAGAGCCCGGCGAGCACAGA-3’。
Recovering PCR amplified products, carrying out double enzyme digestion with pEGFP-N1 vector by using XhoI endonuclease and EcoRI endonuclease respectively to obtain digested PCR amplified products and linearized pEGFP-N1 vector, then connecting by using T4DNA ligase to obtain connection products, converting the connection products into E.coli JM109 competent cells, culturing the connection products in solid LB culture medium at a constant temperature of 37 ℃, picking monoclonal colonies for strain proliferation after inversion culture for 12 hours, extracting plasmids by using a deiotoxin plasmid extraction kit, carrying out double enzyme digestion identification by using XhoI endonuclease and EcoRI endonuclease, and identifying correct plasmids as pEGFP-N1-TRIM5 plasmids.
pEGFP-N1-TRIM5 plasmid was transfected into 3D4/21 cells, observed under a fluorescent microscope, and 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.
3D4/21 cells transfected with pEGFP-N1-TRIM5 plasmid were infected with porcine Sesinkavirus at an inoculum size of MOI=1.0 24h after transfection, cell samples at 6h, 12h, 24h, 36h and 48h after infection were collected, RNA and protein in the cell samples 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 And Western Blot to detect replication of SVA in the cell sample.
The control group was set as follows: the pEGFP-N1 plasmid was transfected into 3D4/21 cells, treated equally and tested for TRIM5 expression and SVA replication in the cells.
2. Experimental results
The results of the digestion identification are shown in FIG. 3, and the results show that: the sizes of the two electrophoresis bands are about 4700bp and 1489bp respectively, which are consistent with the expected result, thus indicating that the construction of the recombinant plasmid is successful.
The observation and detection diagram of the transfection of pEGFP-N1-TRIM5 plasmid into 3D4/21 cells is shown in FIG. 4, wherein a is the fluorescence microscopic observation diagram of the 3D4/21 cells transfected with pEGFP-N1 plasmid; b is a fluorescence microscope view of 3D4/21 cells transfected with pEGFP-N1-TRIM5 plasmid; c is the detection result of qRT-PCR, wherein vector is the detection result of pEGFP-N1 vector; d is the detection result of Western Blot.
The results show that: the plasmid was successfully transfected into 3D4/21 cells, and the expression level of TRIM5 was significantly increased in the transfected cells.
The SVA replication situation of the 3D4/21 cells transfected with pEGFP-N1-TRIM5 plasmid and the 3D4/21 cells transfected with pEGFP-N1 plasmid after infection of porcine Seika virus is shown in FIG. 5, and a is the detection result of qRT-PCR; b is TCID 50 Detecting a result by the method; c is the Western Blot detection result.
The results show that: compared with the control group, after cells transfected with pEGFP-N1-TRIM5 plasmid overexpress TRIM5, the gene copy number and virus titer of SVA are significantly reduced, which indicates that promoting the up-regulation of TRIM5 expression can significantly inhibit replication of porcine Session inner card virus.
Example 3 siRNA for improving replication Capacity of Swine Session inner card Virus
1. Experimental method
Designing a gene sequence (NM_ 001044532.1) of TRIM5 on NCBI as a target gene, wherein the gene sequence is shown in SEQ ID NO:10 and the nucleotide sequence of shTRIM5-F is shown as SEQ ID NO:11, and synthesizing the shTRIM5-R with a nucleotide sequence shown as SEQ ID NO by oligo annealing: 1, and a siRNA as shown in 1.
shTRIM5-F(SEQ ID NO：10)：
5’-GATCCCCGGACGAGGAGAAAGTTATTCTttcaagagaAGAATAACTTTCTCCTCGT CCTTTTTA-3’；
shTRIM5-R(SEQ ID NO：11)：
5’-AGCTTAAAAAGGACGAGGAGAAAGTTATTCTtctcttgaaAGAATAACTTTCTCCT CGTCCGGG-3’。
siRNA(SEQ ID NO：1)：5’-GGACGAGGAGAAAGTTATTC-3’。
The oligo annealing step is: taking SEQ ID NO:10 and the nucleotide sequence of shTRIM5-F is shown as SEQ ID NO:11, 1 mu L of shTRIM5-R is added into 48 mu L of annealing buffer solution respectively, and the mixture is blown and mixed uniformly to carry out annealing reaction: and (3) carrying out 5min at 95 ℃ and 15min at 72 ℃, and naturally cooling to room temperature to obtain the siRNA (SEQ ID NO: 1).
siRNA (SEQ ID NO: 1) and an interference vector (pSuper. Retro. Neo+GFP) are subjected to double digestion respectively by using Hind III endonuclease and EcoR I endonuclease to obtain the digested siRNA and the linearized interference vector, the siRNA and the linearized interference vector are connected by using T4DNA ligase to obtain a connection product 1, the connection product 1 is transformed into E.coli JM109 competent cells, the E.coli JM109 competent cells are placed in LB medium for constant temperature culture at 37 ℃, monoclonal colonies are picked and proliferated, bacterial precipitates are collected, plasmids are extracted by using a deiotoxin kit, and double digestion electrophoresis identification is performed by using Hind III endonuclease and EcoR I endonuclease.
And collecting the plasmid which is identified to be correct by electrophoresis, namely a sh-TRIM5 plasmid, transfecting 3D4/21 cells with the sh-TRIM5, observing under a fluorescence microscope, and respectively carrying out qRT-PCR and Western Blot to detect the expression of the TRIM5 gene in the transfected cells by using TRIM5-F (SEQ ID NO: 4) and TRIM5-R (SEQ ID NO: 5).
Cells were infected with porcine Seika Virus (SVA) at an inoculum size of MOI=1.0 after 24h transfection, cell samples were collected at different time points of 6h, 12h, 24h, 36h and 48h after virus infection, intracellular RNA and protein 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 And Western Blot to detect replication of SVA in the cell sample.
The control group was set as follows: 3D4/21 cells were transfected with an empty plasmid (i.e., pSuper. Retro. Neo+GFP plasmid).
2. Experimental results
The identification result of double enzyme digestion electrophoresis is shown in fig. 6, and the result shows that: the size of the electrophoresis band is about 7100bp, 1130bp and 281bp respectively, meets the requirements, and the recombined nucleotide sequence is shown as SEQ ID NO:1, and the recombinant plasmid of the siRNA shown in the formula 1 is successfully constructed.
The observation and detection diagram of the transfection of the sh-TRIM5 plasmid into 3D4/21 cells is shown in FIG. 7, wherein a is the fluorescence microscope observation diagram of 3D4/21 cells transfected with the sh-NC plasmid; b is a fluorescence microscope observation diagram 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.
The results show that: after the sh-TRIM5 plasmid is transfected into cells, the expression level of TRIM5 in the cells is obviously reduced, which indicates that the sh-TRIM5 plasmid can inhibit the expression of TRIM5 genes.
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 Seika virus is shown in FIG. 8; a is the detection result of qRT-PCR; b is TCID 50 Detecting a result by the method; c is the Western Blot detection result.
The results show that: after the expression level of TRIM5 in the cells was reduced, the copy number and virus titer of SVA increased significantly, indicating that interfering with the expression of TRIM5 gene promoted replication of SVA.
Finally, it should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive of all the embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. The application of an agent for promoting TRIM gene expression in preparing a product for resisting porcine saikovirus.
2. The use according to claim 1, wherein the TRIM gene is TRIM5 gene.
3. The use according to claim 1, wherein the anti-swine saint virus product is a drug that inhibits replication of swine saint virus.
4. Use of an agent that promotes expression of a TRIM protein in the preparation of a product against porcine saikovirus.
5. The use according to claim 4, wherein the TRIM protein is TRIM5 protein.
6. Use of an agent that promotes expression of a TRIM protein and/or TRIM gene in the manufacture of a medicament for treating and/or combating a porcine sai virus infection.
7. The nucleotide sequence is shown in SEQ ID NO:1 in the preparation of a porcine sai virus vaccine.
8. Recombinant nucleotide sequences shown in SEQ ID NO:1 in the preparation of porcine sai virus.
9. A medicament against porcine saikovirus, wherein the medicament inhibits replication of porcine saikovirus by promoting expression of TRIM genes and/or TRIM proteins.
10. The medicament of claim 9, further comprising a pharmaceutically acceptable carrier.
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