Application of pig SAMD5 gene in resisting porcine circovirus type 2 infection

Abstract

The application discloses application of a pig SAMD5 gene in resisting porcine circovirus type 2 infection. SAMD5 The application first discovers and verifies that the pig SAMD5 gene is related to porcine circovirus type 2 infection, and knocking out the pig SAMD5 gene can effectively inhibit replication of porcine circovirus type 2, fills a functional blank in the field of virus infection, and provides a brand-new molecular target for breeding gene editing pigs resistant to porcine circovirus type 2.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and particularly relates to pigs. SAMD5 Application of genes in resistance to porcine circovirus type 2 infection. Background Technology

[0002] Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus in the family Circoviridae. It is one of the smallest viruses known to infect mammals, with a genome of approximately 1.77 kb of single-stranded circular DNA. PCV2 is a major pathogen causing various porcine circovirus-related diseases, including post-weaning multisystemic wasting syndrome (PCVAD), porcine dermatitis-nephropathy syndrome (PDNS), and porcine respiratory disease syndrome (PRDS). First identified as the causative agent of PCVAD in the 1990s, it is now prevalent globally. PCV2 not only directly causes inflammatory damage to tissues and organs but also primarily infects host immune cells, leading to immunosuppression. It is frequently co-infected with other pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae, exacerbating the disease. Although the PCV2 genome is small, its replication is highly dependent on the interaction with various factors within host cells. In recent years, several key host factors involved in PCV2 infection and replication have been identified, such as the four-transmembrane protein CD81 as a functional receptor for viral invasion, the nuclear transport receptor IPO4 promoting viral replication by binding to capsid proteins, and the retinol-binding protein RBP4 limiting viral replication through selective autophagy that degrades viral proteins. Therefore, identifying and utilizing key host factors regulating PCV2 replication from the perspective of host genetics not only helps elucidate the pathogenic mechanisms of the virus but also provides an important theoretical basis for developing host-guided antiviral strategies and screening for disease-resistant breeding targets.

[0003] SAMD5 (Sterile Alpha Motif Domain Containing 5) belongs to a family of proteins containing a sterile alpha motif (SAM) domain. The SAM domain is a conserved protein-protein interaction motif of approximately 70 amino acids, widely found in signal transduction and transcriptional regulatory proteins. SAMD5 contains a SAM domain at its N-terminus, which mediates interactions with the SAM domains of Eph receptor tyrosine kinase families (such as EPHA5, EPHA6, and EPHB1). Although studies have reported aberrant expression of SAMD5 in various tumors, its specific biological function remains unclear, and its function in viral infection-related areas has not yet been reported. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a pig SAMD5 The application of genes in resistance to porcine circovirus type 2 infection, including inhibiting or eliminating porcine circovirus type 2 infection. SAMD5 Gene expression reagents are used to prepare drugs against porcine circovirus type 2 infection, and to inhibit or eliminate porcine circovirus type 2 infection. SAMD5 Gene expression is used in two directions for molecular breeding against porcine circovirus type 2.

[0005] Technical solution: The present invention describes the inhibition or elimination of pig... SAMD5 Application of gene expression reagents in the preparation of drugs against porcine circovirus type 2 infection.

[0006] Preferably, the reagent is selected from any one of siRNA, shRNA, antisense nucleic acid, CRISPR / Cas gene editing system, CRISPRi system, zinc finger nuclease system, and TALEN system.

[0007] Preferably, the application is in the preparation of a drug for the prevention and / or treatment of diseases caused by porcine circovirus type 2 infection; more preferably, the diseases caused by porcine circovirus type 2 infection include, but are not limited to, weaned piglet multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, porcine respiratory disease syndrome, and sow reproductive disorders.

[0008] Preferably, the drug is a drug that inhibits viral replication during the course of porcine circovirus type 2 infection.

[0009] Preferably, the drug is used to inhibit or eliminate pigs SAMD5 The gene expression reagent, as the active ingredient, optionally contains pharmaceutically acceptable excipients.

[0010] Preferably, the dosage form of the drug includes tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, dressings, nebulizing solutions, film-forming agents, implants, microemulsions, liposomes, nanoparticles, oral instant films, sponges, and capsules.

[0011] The invention describes the inhibition or elimination of pigs SAMD5 Application of gene expression in molecular breeding for resistance to porcine circovirus type 2.

[0012] Preferably, the application is for knockout pigs. SAMD5 Genes were used for molecular breeding to resist porcine circovirus type 2.

[0013] Preferably, the knockout is performed using a CRISPR / Cas gene editing system; more preferably, the target sequence for knockout using the CRISPR / Cas gene editing system is shown in SEQ ID NO: 1.

[0014] The invention describes the inhibition or elimination of pigs SAMD5Application of gene expression in the construction of a cell line resistant to porcine circovirus type 2.

[0015] Preferably, the application is for knockout pigs. SAMD5 Genes were used to construct a cell line resistant to porcine circovirus type 2.

[0016] Preferably, the knockout is performed using a CRISPR / Cas gene editing system; more preferably, the target sequence for knockout using the CRISPR / Cas gene editing system is shown in SEQ ID NO: 1.

[0017] The method for inhibiting porcine circovirus type 2 replication according to the present invention comprises the following steps: utilizing a method capable of inhibiting or eliminating host cells or individual pigs... SAMD5 Reagents for gene expression are used to treat host cells or individuals.

[0018] Preferably, the pig SAMD5 The gene includes: the coding sequence corresponding to Gene ID: 100522528 in the NCBI Gene database, or a nucleotide sequence that has at least 80% sequence identity with the coding sequence and has the same function.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to discover and verify the pig SAMD5 The gene is associated with porcine circovirus type 2 infection, and knockout pigs SAMD5 The gene can effectively inhibit the replication of porcine circovirus type 2, filling a gap in [the study / development]. SAMD5 This fills a functional gap in the field of viral infection and provides a novel molecular target for breeding gene-edited pigs resistant to porcine circovirus type 2. Attached Figure Description

[0020] Figure 1 For pigs SAMD5 Porcine circovirus type 2 (PCV2) infection in gene knockout 3D4 / 21 cells and wild-type 3D4 / 21 cells cap Figure showing the results of mRNA relative expression level determination; Figure 2 For pigs SAMD5 Porcine circovirus type 2 (PCV2) infection in gene-overexpressing 3D4 / 21 cells and wild-type 3D4 / 21 cells cap Figure showing the results of mRNA relative expression level determination; Figure 3 For pigs SAMD5 Porcine circovirus type 2 (PCV2) infection in gene knockout PK-15 cells and wild-type PK-15 cells cap Figure showing the results of mRNA relative expression level determination; Figure 4 For pigs SAMD5Porcine circovirus type 2 (PCV2) infection in PK-15 overexpressing cells and wild-type PK-15 cells cap Figure showing the results of mRNA relative expression level determination; Figure 5 For pigs SAMD5 Figure 1. Results of PCV2 Cap protein expression level assay in gene knockout 3D4 / 21 cells and wild-type 3D4 / 21 cells after porcine circovirus type 2 infection. Figure 6 For pigs SAMD5 Figure 1. Results of PCV2 Cap protein expression level assay in overexpressing 3D4 / 21 cells and wild-type 3D4 / 21 cells after infection with porcine circovirus type 2. Figure 7 For pigs SAMD5 Figure 1. Results of PCV2 Cap protein expression level assay in gene knockout PK-15 cells and wild-type PK-15 cells after infection with porcine circovirus type 2. Figure 8 For pigs SAMD5 Figure 1. Results of PCV2 Cap protein expression level assay in PK-15 overexpressing cells and wild-type PK-15 cells after porcine circovirus type 2 infection. Figure 9 For pigs SAMD5 Representative images of indirect immunofluorescence staining of PCV2 Cap protein in 3D4 / 21 cells and wild-type 3D4 / 21 cells after infection with porcine circovirus type 2. Detailed Implementation

[0021] The technical solution of the present invention will be further described below.

[0022] Example 1: Porcine protein containing SAM domain 5 ( SAMD5 Construction of gene knockout vectors and overexpression vectors 1. Reorganization SAMD5 Construction of gene knockout vector For the public SAMD5 A specific sgRNA target sequence as shown in SEQ ID NO: 1 was designed based on the coding sequence (SEQ ID NO: 2) corresponding to the gene sequence (NCBI Gene ID: 100522528). Complementary oligonucleotide chains with BbsI enzyme sticky ends as shown in SEQ ID NO: 3 and 4 were synthesized based on the target sequence. After being mixed in equimolar ratio, the mixture was heated at 95℃ for 5 min and then naturally cooled to room temperature to form a double-stranded DNA insert fragment.

[0023] The pSpCas9(BB)-2A-Puro (pX459) V2.0 vector (Addgene, Plasmid #62988) was linearized by BBSI digestion, then mixed with the aforementioned double-stranded DNA insert fragment, and ligated using T4 DNA ligase at 16°C for 16 hours. The ligation product was then transformed into... E. coli DH5α competent cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated at 37°C for 12 h. After incubation, single colonies were picked and sequenced by Beijing Qingke Biotechnology Co., Ltd. to verify their correctness, yielding recombinant cells. SAMD5 Gene knockout vector pX459-p SAMD5 -KO is used for subsequent experiments.

[0024] 2. Reorganization SAMD5 Construction of gene overexpression vectors Based on as shown in SEQ ID NO: 2 SAMD5 Gene coding sequences, design and synthesis SAMD5 Amplification primers, sequences as follows: SAMD5 -F, the sequence is: 5'-ctagcgtttaaacttaagcttGCCACCATGTGCACCAACATAGTTTACGAGT-3'; SAMD5 -R, the sequence is: 5'-atggtctttgtagtcctcgagGCGGTTCTGTGATGACTTCGG-3'.

[0025] Total RNA was extracted from porcine alveolar macrophages 3D4 / 21 using TRIzol reagent (Thermo Fisher Scientific Inc., catalog number 15596018), and then cDNA was obtained by reverse transcription using the Novizan HiScript II Q RT SuperMix for qPCR kit (catalog number R222). SAMD5 Using the aforementioned cDNA as a template, PCR amplification was performed using Phanta Super-Fidelity DNA Polymerase (catalog number P501) with the following reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 15 s, 35 cycles; final extension at 72℃ for 5 min. After the PCR reaction, the amplification products were verified by 2% agarose gel electrophoresis and recovered using a PCR product recovery kit (OmegaBio-Tek, catalog number D6492). SAMD5CDS nucleic acid fragment.

[0026] pECMV-3×Flag-C (Hunan Fenghui Biotechnology Co., Ltd., product number ZT1018) was digested with HindIII and XhoI, and then combined with the aforementioned... SAMD5 CDS nucleic acid fragments were mixed and ligated using the Novizan ClonExpress II One-Step Cloning Kit (catalog number C112). The ligation product was then transformed into... E. coli DH5α competent cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated at 37°C for 12 h. After incubation, single colonies were picked and sequenced by Beijing Qingke Biotechnology Co., Ltd. to verify their correctness, yielding recombinant cells. SAMD5 gene overexpression vector pECMV- pSAMD5 -3×FLAG.

[0027] Example 2: Pig SAMD5 Construction of gene knockout cell lines Porcine alveolar macrophages 3D4 / 21 and porcine kidney cells PK-15 were seeded into 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator until the cell confluence reached 75±5%. Sartorius jetPRIME was then used to culture the cells. ® DNA / siRNA transfection reagent (catalog number 101000001) was used to prepare transfection kits for pX459-p. SAMD5 The transfection complex of -KO was used for transfection; untransfected cells were set up as a control group.

[0028] Twenty-four hours after transfection, the original medium was replaced with fresh DMEM complete medium containing 2 μg / mL puromycin. Selection continued for 72 hours, with cell status observed every 24 hours and the medium replaced with fresh puromycin-containing complete medium until all untransfected control cells died. Surviving resistant cell confluences were collected for subsequent monoclonal culture. Monoclonal culture was performed using a limiting dilution method: the puromycin-selected resistant cells were trypsinized, resuspended, and counted, then serially diluted to a final concentration of 5 cells / mL. These cells were seeded into 96-well plates at a seeding volume of 100 μL / well, ensuring an average of 0.5 cells per well. Once monoclonal cell clusters formed and filled the well bottoms, the cells were expanded to 24-well plates.

[0029] After culture, cells were collected, washed twice with pre-cooled PBS, and lysed on ice for 30 min using RIPA strong lysis buffer (Beijing Pulilai Gene Technology Co., Ltd., catalog number C1053) containing a mixture of protease inhibitors and phosphatase inhibitors (Shanghai Beyotime Biotechnology Co., Ltd., catalog number P1046). After centrifugation at 12000×g for 15 min at 4℃, the supernatant was collected. The protein concentration was determined by the BCA method and the concentrations of each group were adjusted to be consistent. Then, 5×SDS loading buffer (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 20315ES05) was added according to the ratio. The mixture was denatured in a metal bath at 98℃ for 10 min, followed by SDS-PAGE electrophoresis. The mixture was then transferred to a PVDF membrane and blocked. SAMD5 primary antibody (provided by Yangzhou University, prepared according to the method described by Qiu Yaxuan et al. Construction of FAM50A gene knockout Beta-TC-6 cell line and preparation of polyclonal antibody for FAM50A using CRISPR / Cas9 technology [J]. Journal of Anhui Medical University, 2025, 60(11):2105-2112) or β-Actin primary antibody (Santa) diluted 1:500 was added. Incubate with Cruz (catalog number sc-47778) overnight at 4°C. After rinsing, incubate with HRP-labeled secondary antibody (Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog numbers CW0102 and CW01023) diluted 1:5000 at room temperature for 1 h. Finally, develop and acquire images using ECL chemiluminescence. Cell lines without protein bands at 15-25 kDa are SAMD5-deficient positive monoclonal cell lines, which are passaged and stored for later use.

[0030] Example 3: Pig SAMD5 Effects of genes on porcine circovirus type 2 (PCV2) replication Take the pig obtained in Example 2 SAMD5 Gene knockout 3D4 / 21 cell line (3D4 / 21- SAMD5 KO and wild-type 3D4 / 21 cells (3D4 / 21-Control) were seeded in 6-well plates using DMEM medium containing 10% fetal bovine serum (FBS) and cultured at 37°C in a 5% CO2 incubator until the cell confluence reached 70%. The cells were then inoculated with PCV2 virus solution (PCV2d genotype, provided by Yangzhou University) and the virus was absorbed at 37°C for 1 h to complete the virus infection. The culture medium was then replaced with fresh 2% FBS DMEM medium for further culture.

[0031] Meanwhile, at a cell confluence of 70%, a portion of wild-type 3D4 / 21 cells were transfected with 2 μg of recombinant DNA using Sartorius jetPRIME® DNA / siRNA transfection reagent. SAMD5 gene overexpression vector pECMV- pSAMD5-3×FLAG, transfected for 48 h and then seeded into 12-well plates to obtain overexpression. SAMD5 3D4 / 21 cells of the gene (3D4 / 21- SAMD5 OE The virus was then inoculated with PCV2 virus solution (PCV2d genotype, MOI = 1), and the virus infection was completed by adsorption at 37°C for 1 h. The culture medium was then replaced with fresh 2% FBS DMEM medium for further culture.

[0032] Following the aforementioned experimental procedures, take the pigs obtained in Example 2. SAMD5 PK-15 gene knockout cell line (PK-15- SAMD5 KO and wild-type PK-15 cells (PK-15-Control) were used, and overexpression was prepared based on wild-type PK-15 cells. SAMD5 PK-15 cells (PK-15-) SAMD5 OE ), and infect cells with PCV2.

[0033] 1. Detection of viral gene transcription by qPCR Cells from each group were collected at 12, 24, and 48 h post-infection, and uninfected cells (0 h) were also collected. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific Inc., catalog number 15596018). RNA concentration and purity were measured using a NanoDrop 1000 spectrophotometer. cDNA was then obtained by reverse transcription using the Novizan HiScript II Q RT SuperMix for qPCR kit (catalog number R222) with the primers shown below: PCV2 Cap Upstream primer: 5'-ccaggagggcgttctgact-3'; PCV2 Cap Downstream primer: 5'-cgttaccggctggagaaggaa-3'; β-Actin upstream primer: 5'-ctccatcatgaagtgcgacgt-3'; β-Actin downstream primer: 5'-gtgatctccttctgcatcctgtc-3'. qPCR detection was performed using a quantitative real-time PCR kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number Q311). The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing extension for 30 s, for 40 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. β-Actin was used as an internal control, and Ct values ​​were calculated. -ΔΔCt Method for calculating PCV2 Cap The relative expression level of genes.

[0034] The results are as follows Figure 1-4 As shown, SAMD5Overexpression significantly promoted the expression of the PCV2 cap gene in 3D4 / 21 and PK-15 cells, while SAMD5 Knockout significantly suppressed the expression of the PCV2 cap gene in 3D4 / 21 and PK-15 cells.

[0035] 2. Western blot method for detecting viral protein expression Cells from each group were collected at 12, 24, and 48 h post-infection. Uninfected cells (0 h) were also collected, washed twice with pre-cooled PBS, and proteins were extracted and denatured according to the method described in Example 2. After quantification, the proteins were denatured, transferred to a PVDF membrane and blocked after SDS-PAGE electrophoresis. Then, PCV2 Cap primary antibody (GeneTex, catalog number GTX128121) diluted 1:1000, β-Actin primary antibody diluted 1:500, or Flag primary antibody (Beijing Bomei Biotechnology Co., Ltd., catalog number M185) diluted 1:1000 were added and incubated overnight at 4°C. After rinsing, HRP-labeled secondary antibody diluted 1:5000 was added and incubated at room temperature for 1 h. Finally, the cells were developed and images were acquired by ECL chemiluminescence.

[0036] The results are as follows Figure 5-8 As shown, SAMD5 Overexpression significantly promoted the expression of PCV2 Cap protein in 3D4 / 21 and PK-15 cells, while SAMD5 Knockout significantly inhibited the expression of PCV2 Cap protein in 3D4 / 21 and PK-15 cells.

[0037] 3. Detection of viral protein expression by indirect immunofluorescence assay Following the aforementioned experimental procedures, take the pigs obtained in Example 2. SAMD5 Gene knockout 3D4 / 21 cell lines and wild-type 3D4 / 21 cells were prepared, and overexpression was generated based on wild-type 3D4 / 21 cells. SAMD5 3D4 / 21 cells were seeded into 12-well cell culture plates with sterile circular slides placed at the bottom. When the cells adhered and grew to approximately 70% confluence, they were inoculated with PCV2 virus solution (PCV2d genotype, MOI = 1). Virus infection was completed at 37°C for 1 h, and then the medium was replaced with fresh 2% FBS DMEM for further culture.

[0038] After culturing for 48 h, the culture supernatant was discarded, and the cells were washed three times with pre-cooled PBS. Then, 4% paraformaldehyde solution was added and the cells were fixed at room temperature for 15 min. The cells were washed three times again with PBS, and incubated for 10 min at room temperature with PBS containing 0.1% Triton X-100 (permeabilization solution). Blocking was then performed for 1 h at room temperature with PBS containing 5% bovine serum albumin (BSA). After blocking, PCV2 Cap primary antibody diluted 1:200 was added, and the cells were incubated overnight at 4°C in a humidified chamber. The cells were washed three times with PBS containing 0.05% (v / v) Tween-20 for 5 min each time. Then, fluorescent secondary antibody diluted 1:500 (Proteintech, catalog number SA00013-2) was added, and the cells were incubated at room temperature in the dark for 1 h. After incubation, the cells were washed thoroughly again, and the nuclei were counterstained with DAPI staining solution. The cells were then mounted with anti-fluorescence quenching mounting medium and observed and images were acquired using a laser scanning confocal microscope.

[0039] The results are as follows Figure 9 As shown, compared with the control group, SAMD5 In cells overexpressing PCV2 Cap protein, the positive signal of PCV2 Cap protein was significantly increased, fluorescence intensity was enhanced, and the proportion of infected cells increased; while SAMD5 The positive signal of PCV2 Cap protein was significantly reduced in knockout cells.

[0040] The above results indicate that SAMD5 Positive regulatory role in PCV2 infection and replication, knockout pigs SAMD5 The gene can significantly inhibit the replication of porcine circovirus type 2.

Claims

1. A method for inhibiting or eliminating pig... SAMD5 Application of gene expression reagents in the preparation of drugs against porcine circovirus type 2 infection.

2. The application according to claim 1, characterized in that, The reagents are selected from any one of siRNA, shRNA, antisense nucleic acid, CRISPR / Cas gene editing system, CRISPRi system, zinc finger nuclease system, and TALEN system.

3. The application according to claim 1, characterized in that, The drug is used to inhibit viral replication during the course of porcine circovirus type 2 infection.

4. The application according to claim 1, characterized in that, The drug is used to inhibit or eliminate pigs SAMD5 The gene expression reagent, as the active ingredient, optionally contains pharmaceutically acceptable excipients.

5. A method for inhibiting or eliminating pig... SAMD5 Application of gene expression in molecular breeding for resistance to porcine circovirus type 2.

6. The application according to claim 5, characterized in that, The application is for knockout pigs. SAMD5 Genes were used for molecular breeding to resist porcine circovirus type 2.

7. The application according to claim 6, characterized in that, The knockout was performed using the CRISPR / Cas gene editing system.

8. The application according to claim 7, characterized in that, The target sequence for knockout based on the CRISPR / Cas gene editing system is shown in SEQ ID NO:

1.

9. A method for inhibiting or eliminating pig... SAMD5 Application of gene expression in the construction of a cell line resistant to porcine circovirus type 2.

10. The application according to claim 1, 6, or 9, characterized in that, The pig SAMD5 The gene includes: the coding sequence corresponding to Gene ID: 100522528 in the NCBI Gene database, or a nucleotide sequence that has at least 80% sequence identity with the coding sequence and has the same function.

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