Application of DDIT3 gene as target spot in regulation and control of bovine coronavirus replication

Abstract

The invention discloses application of a DDIT3 gene as a target spot in regulation and control of bovine coronavirus replication. Experiments find that regulation and control of the expression level of the DDIT3 gene can influence the replication level of the BCoV, when the expression of the DDIT3 gene in a host cell is inhibited, the replication of the BCoV can be promoted and the titer of the virus can be improved, and when the DDIT3 is over-expressed on the host cell, the replication of the BCoV can be inhibited and the titer of the virus can be reduced. Experimental results show that the DDIT3 gene can be used as a novel target for developing BCoV antiviral drugs, and a tool and a material are provided for researching a molecular mechanism of the DDIT3 gene for regulating and controlling pathogenic microorganism replication in cells.

Description

Technical Field

[0001] This invention belongs to the fields of animal genetic engineering and molecular biology, and relates to the screening, verification and application of bovine coronavirus disease resistance genes. Background Technology

[0002] Bovine coronavirus (BCoV) is a single-stranded positive-sense RNA virus with irregularly shaped particles, ranging from 65 to 210 nm in diameter. Belonging to the family Coronaviridae and the genus Betacoronavirus, BCoV has a genome size of 27–30 kb. The BCoV genome includes untranslated regions (5'-UTR) and 3'-UTRs at both ends. The 5' end has a methylated cap-like structure, and the 3' end has a poly A tail. It contains two open reading frames (ORF1a and ORF1b) encoding 16 non-structural proteins that regulate RNA synthesis and modification, and five structural proteins: nucleocapsid protein (N), membrane protein (M), hemagglutinin-esterase protein (HE), spike protein (S), and minor membrane protein (E). BCoV infection typically occurs in autumn and winter and has become an epidemic trend. Although the mortality rate in adult cattle infected with the virus is low, it causes a significant decrease in milk production and body weight. To date, all BCoV isolates belong to a single serotype, but there are two to three subtypes, and the antigenicity varies among different isolates.

[0003] Besides infecting cattle, bovine coronaviruses also have the potential for cross-species transmission. Studies have shown that bovine coronaviruses share up to 96% genomic homology with human coronavirus OC43. Furthermore, a study analyzed a human enteric coronavirus named HEC 4408 isolated from fecal samples of children with acute diarrhea, and its genome analysis showed that HEC 4408 is more closely related to bovine coronaviruses in terms of genetic evolution than HEC 4408 is related to human coronaviruses. Additionally, HEC 4408 and bovine coronaviruses... S Gene, HE The gene homology is as high as 99%, indicating that HEC 4408 is a bovine coronavirus variant that infects humans.

[0004] Regarding the prevention and control of bovine coronavirus infection, there are reports of vaccine trials (see "Prokaryotic Expression of Bovine Coronavirus N Protein and Preliminary Evaluation of Immunogenicity in Mice. Chinese Journal of Veterinary Medicine, 2024, 44(12)2540-2548."). In addition, there are also reports surrounding the virus... HE Reports on gene-based research into the replication and transmission mechanisms of bovine coronaviruses (see "... HE The effect of gene receptor binding domain variation on bovine coronavirus infection. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56 (03):1336-1343.

[0005] DNA damage-induced transcription factor 3 (DDIT3), also known as CHOP or GADD153, is a transcription factor produced by DNA damage-induced transcription factor 3 (DDIT3). DDIT3 A ubiquitous transcription factor encoded by genes, it can form heterodimers with other members of the C / EBP and Jun / Fos families and is an important component of the stress-induced transcriptional network. Under normal conditions... DDIT3 DDIT3 is expressed at low levels. When cells are subjected to DNA damage, endoplasmic reticulum stress, hypoxia, and starvation, transcription and translation are activated, leading to the accumulation of DDIT3 protein and inducing apoptosis. The biological activity of DDIT3 is also regulated by phosphorylation by kinases such as p38. Unlike other C / EBP transcription factors, DDIT3 cannot form homodimers. Instead, DDIT3 acts as a major negative regulator; the activity of other proteins in its family can be attenuated by the heterodimer of DDIT3, which can exert transcriptional regulatory functions by binding to DNA. Although increasing research indicates that DDIT3 plays an important role in endoplasmic reticulum stress-mediated apoptosis and autophagy, no evidence has yet been found that it can transfer host cells to DDIT3. DDIT3 Reports of genes acting as resistance genes for bovine coronavirus. Summary of the Invention

[0006] The purpose of this invention is to provide DDIT3 Application of genes as targets in regulating bovine coronavirus replication.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, a method for preparing high-titer bovine coronavirus is provided, comprising the following steps: Knockdown human cells DDIT3 Genes were used to obtain knockdown cells (e.g., interference cells); the knockdown cells obtained by bovine coronavirus infection were cultured, and the cell culture supernatant was separated after at least 48 h of culture to obtain high-titer bovine coronavirus.

[0008] Preferably, the knockdown employs RNA interference technology, that is, targeting human... DDIT3 Genetically engineered small interfering RNA (siRNA) is transfected into human cells to obtain interfering cells.

[0009] Preferably, the sequence of the siRNA is shown in SEQ.ID.NO.2.

[0010] Preferably, the human cells are human ileocecal cancer cells (e.g., HCT-8).

[0011] Secondly, a cell model that promotes bovine coronavirus infection is provided, which is achieved by using methods to inhibit endogenous...DDIT3 It is made by treating the above-mentioned human cells with gene expression agents or reagents.

[0012] Preferably, the formulation includes DDIT3 Inhibitors of the gene / DDIT3 protein (such as those targeting humans mentioned above) DDIT3 (Gene-designed siRNA).

[0013] Preferably, the reagent includes DDIT3 Inhibitors of the gene / DDIT3 protein (e.g., human) DDIT3 Drug molecules that target genes and can reduce the expression level of DDIT3 protein.

[0014] Preferably, the cell model is a viral amplification cell (e.g., knocked-down) capable of increasing intracellular viral replication levels and viral titers in cell culture supernatant after infection with bovine coronavirus. DDIT3 Human cells expressing genes).

[0015] Preferably, the cell model is specifically achieved by interfering with human cells. DDIT3 Gene expression (e.g., through transfection of the above-mentioned human targets) DDIT3 The cells, constructed using genetically engineered siRNA, can be inoculated with the virus and cultured to obtain high-titer bovine coronavirus. These cells, which can improve viral amplification efficiency, are helpful for better studying the pathogenic mechanism of bovine coronavirus and for more efficient amplification of bovine coronavirus vaccine strains.

[0016] Thirdly, it provides a method for knocking down people. DDIT3 Interfering RNA for genes, including siRNA with a sequence as shown in SEQ ID NO.2.

[0017] Fourthly, providing... DDIT3 Application of gene / DDIT3 protein-targeting activators in the preparation of drugs (e.g., antiviral drugs) for the prevention and / or treatment of bovine coronavirus infection.

[0018] Preferably, the drug is used to promote human... DDIT3 Gene expression preparations or reagents.

[0019] Preferably, the person DDIT3 The gene was selected from NCBI accession number NC_000012.12. DDIT3 Genes, or the DDIT3 protein-coding sequence as shown in SEQ.ID.NO.1.

[0020] Preferably, the formulation comprises a human DDIT3 protein overexpression vector (e.g., pcDNA3.1-DDIT3), which, upon introduction (e.g., transfection) into the aforementioned human cells, activates the cells by increasing the expression level of DDIT3 protein in the corresponding cells. DDIT3 The gene (i.e., activating the expression of the DDIT3 protein) inhibits the replication of bovine coronavirus.

[0021] Preferably, the reagent comprises human... DDIT3 Drug molecules that target genes and can increase the expression level of DDIT3 protein.

[0022] Fifthly, a cell model for resisting bovine coronavirus infection is provided, which is prepared by treating the aforementioned human cells with an agent or preparation for promoting the expression of the human DDIT3 gene.

[0023] Preferably, the formulation comprises a human DDIT3 protein overexpression vector (e.g., pcDNA3.1-DDIT3).

[0024] Preferably, the reagent comprises human... DDIT3 Drug molecules that target genes and can increase the expression level of DDIT3 protein.

[0025] The beneficial effects of this invention are reflected in: This invention, through the construction of a DDIT3 overexpression vector, is the first to discover that increasing the expression level of DDIT3 in human cells (such as HCT-8 cells) can reduce the replication level of bovine coronavirus. This finding suggests that... DDIT3 Activators of the gene (or DDIT3 protein) can be used in the development of novel antiviral drugs against bovine coronavirus infection. Additionally, this invention utilizes RNA interference technology to knock down human cells. DDIT3 Gene, first discovery of inhibition DDIT3 The expression of this gene can promote the replication of bovine coronavirus within cells and increase the viral titer in cell culture supernatant. This finding suggests that... DDIT3 Gene inhibitors can be used to obtain high titers of bovine coronavirus.

[0026] This invention is based on the change in DDIT3 expression levels in human cells (such as HCT-8 cells) infected with bovine coronavirus, and knocks it down in the corresponding host cells. DDIT3 Experiments on gene and overexpression of DDIT3 not only rely on experimental results DDIT3 Genetic identification revealed it to be a resistance gene against bovine coronavirus (i.e., promoting immunity). DDIT3 Gene expression inhibits the replication of bovine coronavirus, and when DDIT3When gene expression was suppressed, the replication level of bovine coronavirus was significantly increased, and viral load was also increased. Furthermore, it was found that the virus could be effectively suppressed. DDIT3 Interfering RNA for gene expression (which also acquired the ability to promote) DDIT3 (DDIT3 overexpression plasmid for gene expression), and for research DDIT3 This study provides important tools and materials (including highly efficient viral amplification cells and high-titer viruses) for understanding the molecular mechanisms by which genes regulate the replication of pathogenic microorganisms within cells and for the preparation of bovine coronavirus vaccines. Attached Figure Description

[0027] Figure 1 Results of DDIT3 expression levels in HCT-8 cells after BCoV infection: A shows the expression levels of DDIT3 protein in HCT-8 cells detected by Western blot at 12 h, 24 h, 36 h, and 48 h post-BCoV infection (GAPDH was used as an internal control); B shows the expression levels of DDIT3 protein in HCT-8 cells at different time points post-BCoV infection detected by real-time quantitative PCR. DDIT3 Gene mRNA levels (*** indicates relative to the BCoV-uninfected group, i.e., the MOCK group) DDIT3 Gene expression level p <0.005, ** indicates that compared to the BCoV-uninfected group, i.e., the MOCK group. DDIT3 Gene expression level p <0.01, ns indicates relative to the BCoV uninfected group, i.e., the MOCK group. DDIT3 Gene expression level p >0.05).

[0028] Figure 2 Results of DDIT3 overexpression vector construction and overexpression effect evaluation: A is... DDIT3 Identification of PCR products of genes (lane M is DL 5000 plus DNA Marker, lane "negative" is template-free negative control, lane 1 is...) DDIT3 (PCR product of the gene); B is pcDNA3.1-DDIT3 plasmid digestion identification (lane M is DL 5000 plus DNA Marker, lane "plasmid" is the undigested plasmid, lane "double digestion" is the recombinant plasmid). Hind Ⅲ / Xho I represents double enzyme digestion); C represents Western blot detection of DDIT3 protein expression level in HCT-8 cells (GAPDH is the internal control, pcDNA3.1 group is the negative control of the empty vector in the overexpression group, and pcDNA3.1-DDIT3 group, i.e., the overexpression group, is the cell transfected with pcDNA3.1-DDIT3 plasmid).

[0029] Figure 3Results of the effect of DDIT3 overexpression on BCoV replication: A shows the effect of real-time quantitative PCR detection of bovine coronavirus in HCT-8 cells. N Gene (BCoV) N (Gene) mRNA level (BCoV group: cells infected with BCoV only; pcDNA3.1+BCoV group: cells transfected with empty vector and then infected with bovine coronavirus; pcDNA3.1-DDIT3+BCoV group: cells transfected with constructed overexpression vector and then infected with bovine coronavirus; GAPDH is an internal control; *** indicates relative to BCoV in the BCoV group) N Gene expression level p <0.005, ns indicates relative to BCoV in the BCoV group N Gene expression level p >0.05); B represents the Western blot detection of BCoV replication level (BCoV N protein expression level) and DDIT3 protein expression level in HCT-8 cells (GAPDH as internal control); C represents the detection of the half-maximal tissue culture infectious dose (TCID) of virus in the HCT-8 cell culture supernatant. 50 The BCoV group consists of cells infected with BCoV only; the pcDNA3.1+BCoV group consists of cells transfected with an empty vector and then infected with bovine coronavirus; and the pcDNA3.1-DDIT3+BCoV group consists of cells transfected with a constructed overexpression vector and then infected with bovine coronavirus. ** indicates the viral TCID relative to the BCoV group. 50 level p <0.01, ns indicates the viral TCID relative to the BCoV group. 50 level p >0.05.

[0030] Figure 4 for DDIT3 Identification results of gene interference RNA: Western blot detection of HCT-8 cell transfection DDIT3 DDIT3 protein expression level after gene interference RNA transfection (si-NC group: cells transfected with negative control siRNA; si-DDIT3 group: cells transfected with...) DDIT3 (The siRNA of the gene is used in cells, with GAPDH as an internal control).

[0031] Figure 5 For interference DDIT3 Results of the effect of gene expression on BCoV replication: A shows the results of real-time quantitative PCR detection of the bovine coronavirus N gene (BCoV) in HCT-8 cells. N(Gene) mRNA level (BCoV group: cells infected with BCoV only; si-NC+BCoV group: cells transfected with negative control siRNA and then infected with BCoV; si-DDIT3+BCoV group: cells transfected with...) DDIT3 Cells infected with BCoV were then infected with siRNA of the gene, with GAPDH serving as an internal control; *** indicates BCoV relative to the BCoV group. N Gene expression level p <0.005, ns indicates relative to BCoV in the BCoV group N Gene expression level p >0.05); B represents the Western blot detection of BCoV replication level (BCoV N protein expression level) and DDIT3 protein expression level in HCT-8 cells (GAPDH as internal control); C represents the detection of the half-maximal tissue culture infectious dose (TCID) of virus in the HCT-8 cell culture supernatant. 50 The BCoV group consisted of cells infected with BCoV only, the si-NC+BCoV group consisted of cells transfected with negative control siRNA and then infected with BCoV, and the si-DDIT3+BCoV group consisted of cells transfected with... DDIT3 Cells infected with BCoV were then infected with siRNA of the gene. * indicates viral TCID relative to the BCoV group. 50 level p <0.05, ns represents the viral TCID relative to the BCoV group. 50 level p >0.05. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0033] 1. DDIT3 expression level after HCT-8 infection Materials and reagents: HCT-8 cells were preserved by the Veterinary Public Health Team of the College of Veterinary Medicine, Northwest A&F University (or could be purchased); Primary antibodies: DDIT3 antibody (rabbit) was purchased from Shanghai Abmart, BCoV N protein antibody (mouse) was kindly provided by Professor Yin Xin of Harbin Veterinary Research Institute, and GAPDH antibody (mouse) was purchased from Shanghai Abmart; BCoV strain was provided by the Veterinary Public Health and Livestock Product Safety Laboratory of Northwest A&F University (refer to "The Influence of HE Gene Receptor Binding Domain Variation on Bovine Coronavirus Infectivity. Northwest A&F University, 2024."); Secondary antibody: Horseradish peroxide (HRP) labeled goat anti-mouse IgG antibody was purchased from Shanghai Diyi Biotechnology Co., Ltd.; mRNA reverse transcription and quantification kits and qPCR kits were purchased from Beijing TransGen Biotech Co., Ltd.; PVDF membranes were purchased from Immobilon Biotechnology Co., Ltd.; ECL chromogenic solution was purchased from Beijing Dining Biotechnology Co., Ltd.; all other routine reagents were of analytical grade.

[0034] After HCT-8 cells were infected with bovine coronavirus (BCoV) with an MOI of 10, cells were collected at 0 h (uninfected MOCK), 12 h, 24 h, 36 h, and 48 h (specifically, the culture medium was removed, and the cells were washed). Cells were lysed with RIPA lysis buffer containing a protease inhibitor, lysed on ice for 15 min, then loaded with loading buffer and boiled for 10 min. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. After blocking with 5% milk powder or 5% BSA for 2 h, the membrane was treated with primary and secondary antibodies before imaging. Viral replication (BCoV N protein expression) and DDIT3 protein expression were shown below. Figure 1 As shown in Figure A, the results indicate that bovine coronavirus infection leads to increased expression of the DDIT3 protein in its human host cells.

[0035] After HCT-8 cells were infected with bovine coronavirus with an MOI of 10, cells were collected at 0 h, 12 h, 24 h, 36 h, and 48 h. Total RNA was extracted from the collected cells using the TRIZOL precipitation method. The extracted total RNA was then reverse transcribed into cDNA. Specifically, the reverse transcription reaction system (Table 1) was placed in a PCR instrument and incubated at 42°C for 30 min. After incubation, the reverse transcriptase was inactivated by heating at 85°C for 5 s.

[0036] Table 1. Reverse transcription system for synthesizing cDNA

[0037] Using cDNA obtained from reverse transcription as a template, intracellular cDNA was detected using qPCR primers (Table 3). DDIT3The gene mRNA level was specifically determined by placing the qPCR reaction system (Table 2) in a PCR instrument and pre-denaturing it at 94℃ for 30 s, followed by 40 cycles of (94℃, 5 s; 58℃, 15 s; 72℃, 10 s).

[0038] Real-time quantitative PCR results showed that bovine coronavirus infection caused infection in human host cells. DDIT3 Increased gene mRNA levels ( Figure 1 B).

[0039] Table 2. qPCR reaction system

[0040] Table 3. qPCR primers

[0041] 2. Construction of DDIT3 overexpression vector Materials and reagents: HCT-8 cells were preserved by the Veterinary Public Health Team of the College of Veterinary Medicine, Northwest A&F University (or could be purchased); plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. Xho I enzyme, Hind Enzyme III was purchased from TaKaRa, Phanta Max high-fidelity enzyme was purchased from Nanjing Novizan Biotechnology Co., Ltd., pcDNA3.1 vector (trade name pcDNA3.1(+)) was purchased from Beijing Qingke Biotechnology Co., Ltd., mRNA reverse transcription and quantification kit was purchased from Beijing TransGen Biotech Co., Ltd., and other routine reagents were of analytical grade.

[0042] According to the information published by the NCBI DDIT3 Gene (NM_001414991.1), designed to amplify DDIT3 Primers for the gene coding sequence (Table 4) were synthesized by Beijing Qingke Biotechnology Co., Ltd., and their sequences are as follows: Upstream primer DDIT3-F: 5'-CCAAGCTTatggcagctgagtcattgc-3'; Downstream primer DDIT3-R: 5'-CCCTCGAGtcatgcttggtgcagattcac-3'.

[0043] Table 4. DDIT3 gene

[0044] Total RNA was extracted from uninfected BCoV HCT-8 cells using the TRIZOL precipitation method, and then the extracted total RNA was reverse transcribed into cDNA (reverse transcription reaction system is shown in Table 1). The target gene was then amplified by PCR using the cDNA as a template, and the PCR products were subjected to agarose gel electrophoresis. The amplification system is shown in Table 5, and the reaction procedure is shown in Table 6. The agarose gel containing the PCR product was excised under blue light, and the PCR product containing the target gene was recovered using a DNA purification and recovery kit. Figure 2 A).

[0045] Using restriction endonucleases Hind III and Xho I. The PCR product and pcDNA3.1 vector were double-digested with enzymes, as shown in Table 7. The reaction conditions were: 37℃ water bath for 2 h. Then, the digested products were electrophoresed on a 1% agarose gel. The agarose gel containing the target gene and vector backbone was excised under blue light and recovered using a DNA purification and recovery kit.

[0046] The ligation system was prepared using the recovered target gene and vector backbone (Table 8), and then the ligation system was placed in a 16°C metal bath for 10 h for ligation.

[0047] Competent E. coli were placed on ice and thawed. All ligation products were added, and the mixture was gently pipetted to mix. The mixture was then incubated on ice for 15 min. Afterward, the cells were heat-activated in a 42°C water bath for 50 s, then quickly transferred to an ice bath and incubated for 2 min. 500 μL of antibiotic-free LB broth was added. 100 μL of the bacterial culture was evenly spread onto LB solid medium containing ampicillin. The cells were incubated upside down at 37°C for 10 h. Single colonies were then picked using an inoculation loop for culture and PCR identification.

[0048] Positive bacteria were cultured on LB liquid medium with ampicillin resistance, and plasmids were extracted for restriction enzyme digestion identification. Specifically, recombinant plasmids were extracted using an endotoxin-free plasmid extraction kit, and then restriction endonucleases were used. Xho I and Hind III. Double digestion was performed (digestion system as shown in Table 7), and the results are as follows: Figure 2 As shown in B.

[0049] The positive bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for recombinant plasmid sequencing. After sequence alignment, the eukaryotic expression plasmid pcDNA3.1-DDIT3 was obtained, and the corresponding positive bacteria were preserved at -80℃ using the glycerol preservation method.

[0050] Table 5. PCR reaction system

[0051] Table 6. PCR reaction procedure

[0052] Table 7. Enzyme digestion system

[0053] Table 8. Connection System

[0054] 3. Verify the intracellular expression of the DDIT3 overexpression vector. Materials and reagents: HCT-8 cells were preserved by the Veterinary Public Health Team of the College of Veterinary Medicine, Northwest A&F University (or could be purchased); Primary antibodies: DDIT3 antibody (rabbit-derived) and GAPDH antibody (mouse-derived) were purchased from Shanghai Abmart; Secondary antibodies: Horseradish peroxide (HRP)-labeled goat anti-mouse IgG antibody was purchased from Shanghai Diyi Biotechnology Co., Ltd.; PVDF membrane was purchased from Immobilon Biotechnology Co., Ltd.; ECL chromogenic solution was purchased from Beijing Dining Biotechnology Co., Ltd.; TurboFect was purchased from Thermo Fisher Scientific Co., Ltd.; All other routine reagents were of analytical grade.

[0055] HCT-8 cells were cultured to 60% confluency, then digested with trypsin, mixed with RPMI 1640 medium containing 10% FBS, and seeded into 12-well plates. When the cell density reached 70%–90%, plasmid transfection was performed. Before transfection, the old culture medium was discarded, the cells were washed once with PBS, and 500 μL of RPMI 1640 medium containing 2% FBS was added. The pcDNA3.1-DDIT3 plasmid, transfection reagent (plasmid to TurboFect transfection reagent ratio of 1:1.3 or 1:1.5 (μg / μL), plasmid dosage 1.5 μg / well), and 100 μL of serum-free Opti-MEM I medium were added to a 1.5 mL RNase-free centrifuge tube. After mixing thoroughly, the mixture was allowed to stand for 25 min to prepare the transfection complex. This complex was then added dropwise to the culture wells. The pcDNA3.1 vector was processed in the same manner. The culture plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 h.

[0056] Cells were collected after 24 h of culture and lysed with RIPA lysis buffer containing a protease inhibitor. After lysis on ice for 15 min, loading buffer was added and the cells were boiled for 10 min. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. The membrane was blocked with 5% milk powder or 5% BSA for 2 h, then coated with primary and secondary antibodies before development. DDIT3 protein expression was as follows: Figure 2 As shown in Figure C, the results indicate that the pcDNA3.1-DDIT3 plasmid achieved overexpression of the DDIT3 protein in human host cells of bovine coronavirus.

[0057] 4. Over-expressing oneself DDIT3 The effect of genes on BCoV replication Materials and reagents: HCT-8 cells were preserved by the Veterinary Public Health Team of the College of Veterinary Medicine, Northwest A&F University (or could be purchased); the BCoV strain was provided by the Veterinary Public Health and Livestock Product Safety Laboratory of Northwest A&F University (refer to "The Influence of HE Gene Receptor Binding Domain Variation on Bovine Coronavirus Infectivity. Northwest A&F University, 2024."); TurboFect was purchased from Thermo Fisher Scientific; primary antibodies: DDIT3 antibody (mouse source) was purchased from Shanghai Abmart, BCoV N protein antibody (mouse source) was kindly provided by Professor Yin Xin of Harbin Veterinary Research Institute, and GAPDH (mouse source) was purchased from Shanghai Abmart; secondary antibody: horseradish peroxide (HRP) labeled goat anti-mouse IgG antibody was purchased from Shanghai Diyi Biotechnology Co., Ltd.; mRNA reverse transcription and quantification kits and qPCR kits were purchased from Beijing TransGen Biotech Co., Ltd.; PVDF membranes were purchased from Immobilon Biotechnology Co., Ltd.; ECL chromogenic solution was purchased from Beijing Dining Biotechnology Co., Ltd.; all other routine reagents were of analytical grade.

[0058] HCT-8 cells were cultured to 60% density, digested with trypsin, and resuspended in RPMI 1640 medium containing 10% FBS. Cell counts were then performed. 2 μg of pcDNA3.1-DDIT3 plasmid was added to 1×10⁻⁶ cells. 5 Cells were transfected at a ratio of 100 μL per cell in culture plates using liposomes (i.e., TurboFect transfection reagent). Cell groups were prepared by transfecting empty vector (pcDNA3.1 vector), virus-infected cells (without plasmid transfection), and normal cells (without plasmid transfection and virus infection). After transfection, the culture plates were incubated at 37 °C in a 5% CO2 incubator for 48 h, and then infected with bovine coronavirus (MOI=10). After 48 h, bovine coronavirus replication (mRNA and protein expression) levels were measured, and viral TCID in the cell culture supernatant was also detected. 50 .

[0059] mRNA level: Cells were collected 48 h after infection with bovine coronavirus. Total RNA was extracted from the collected cells using the TRIZOL precipitation method. The extracted total RNA was then reverse transcribed into cDNA. Specifically, the reverse transcription reaction system (refer to Table 1) was incubated in a PCR instrument at 42°C for 30 min, followed by heating at 85°C for 5 s to inactivate the reverse transcriptase. The cDNA obtained from reverse transcription was used as a template, and intracellular BCoV was detected using qPCR primers (Table 9). NThe mRNA level was determined by pre-denaturing the qPCR reaction system (refer to Table 2) at 94℃ for 30 s in a PCR instrument, followed by 40 cycles of (94℃, 5 s; 58℃, 15 s; 72℃, 10 s). Real-time quantitative PCR results showed that cells transfected with pcDNA3.1-DDIT3 plasmid showed increased mRNA levels 48 h after bovine coronavirus infection with BCoV. N Gene mRNA levels are suppressed ( Figure 3 A). This result suggests that the intracellular bovine coronavirus replication level varies with... DDIT3 Gene expression increases and decreases.

[0060] Table 9. qPCR primers

[0061] Protein expression levels: Cells were collected 48 h after infection with bovine coronavirus. Cells were lysed with RIPA lysis buffer containing a protease inhibitor, lysed on ice for 15 min, then loaded with loading buffer and boiled for 10 min. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. After blocking with 5% milk powder or 5% BSA for 2 h, the membrane was treated with primary and secondary antibodies before development. The expression levels of DDIT3 and BCoV N proteins in cells are shown below. Figure 3 As shown in Figure B, the expression level of bovine coronavirus protein decreased after overexpression of DDIT3.

[0062] TCID 50 Experiment: Cells infected with the pcDNA3.1 vector, pcDNA3.1-DDIT3 plasmid, and untransfected cells were collected and inoculated for 48 h. The supernatant was then collected and plated one day in advance. HCT-8 cells in logarithmic growth phase were resuspended and the cell concentration was adjusted to 8 × 10⁶ cells / year. 4 Cells / mL were evenly inoculated into 96-well plates, with 8 replicates for each viral concentration gradient. When the cell density reached approximately 80%, the cell culture medium was discarded. The cell culture supernatant collected after inoculation was serially diluted with RPMI 1640 medium to a 10-10 factor. -1 Up to 10 -10 Cell culture supernatant was added to each well of a 96-well plate at a rate of 100 μL, while an equal volume of RPMI 1640 medium was added to the blank control wells. After 2 hours of virus adsorption, the medium was replaced with RPMI 1640 medium containing 2% FBS. Cell cytotoxicity (CPE) was observed daily. Once the cell condition in each well stabilized and the number of diseased wells no longer increased, the viral titer (TCID) of the amplified bovine coronavirus was calculated using the Reed-Muench method. 50 ), through TCID 50 The results of detecting viral titers in cell culture supernatants are as follows: As shown in Figure C, compared to cells infected with BCoV only, overexpression of DDIT3 followed by infection with BCoV significantly reduced the viral titer of BCoV in the cell culture supernatant.

[0063] 5. People Figure 3 Evaluation of the effectiveness of gene-interfering RNA Materials and reagents: HCT-8 cells were preserved by the Veterinary Public Health Team of the College of Veterinary Medicine, Northwest A&F University (or could be purchased); Primary antibodies: DDIT3 antibody (rabbit-derived) and GAPDH (mouse-derived) were purchased from Shanghai Abmart; Secondary antibodies: Horseradish peroxide (HRP)-labeled goat anti-mouse IgG antibody was purchased from Shanghai Diyi Biotechnology Co., Ltd.; PVDF membrane was purchased from Immobilon Biotechnology Co., Ltd.; ECL chromogenic solution was purchased from Beijing Dining Biotechnology Co., Ltd.; TurboFect was purchased from Thermo Fisher Scientific Co., Ltd.; All other routine reagents were of analytical grade.

[0064] First, the design is targeted at people. DDIT3 According to the design principles of siRNA, the ideal siRNA for gene expression interference should have a GC content between 35% and 55%, a length of 19 to 25 nucleotides (lengths greater than 30 nucleotides may lead to non-specific silencing), and should avoid continuous single bases, inverted repeat sequences, and GG structures as much as possible (because siRNA can be cleaved by ribonucleases at the G base of a single strand). The final design will be as follows: DDIT3 Experiments were conducted using gene siRNA (si-DDIT3 for short): si-DDIT3 sense (ie SEQ.ID.NO.2): 5'-CUGACUACCCUCUCUCACUAG-3' si-DDIT3 anti-sense (i.e. SEQ.ID.NO.3): 5'-CUAGUGAGAGAGGGUAGUCAG-3'.

[0065] The negative control siRNA (si-NC) used in the experiment is as follows: si-NC sense: 5'- AAAA -3' si-NC anti-sense: 5'-UUUU-3'.

[0066] The cultured HCT-8 cells were digested and counted beforehand, and then diluted to 2×10⁶ cells / mL with RPMI 1640 medium containing 10% FBS. 4Add 1 mL of cell suspension to each well of the culture plate at a concentration of 1 cell / mL. Transfection can be performed when the cell density reaches 50%. Use TurboFect transfection reagent to transfect the negative control siRNA, DDIT3 The gene siRNA was transfected into HCT-8 cells (siRNA to TurboFect transfection reagent concentration 1:1.3). After transfection, the culture plates were placed in a 37℃, 5% CO2 incubator. After 48 h, the cells were collected and lysed with RIPA lysis buffer containing protease inhibitors. After lysis on ice for 15 min, loading buffer was added and the cells were boiled for 10 min. SDS-PAGE was then performed and the cells were transferred to PVDF membranes. After blocking with 5% milk powder or 5% BSA for 2 h, the cells were coated with primary and secondary antibodies and then developed. Western blot results showed that si-DDIT3 transfected into the cells had a significant effect on cell function. DDIT3 The gene has a high interference efficiency ( DDIT3 ).

[0067] 6. Interference Figure 4 Effects of gene expression on bovine coronavirus replication Materials and reagents: HCT-8 cells were preserved by the Veterinary Public Health Team of the College of Veterinary Medicine, Northwest A&F University (or could be purchased); the BCoV strain was provided by the Veterinary Public Health and Livestock Product Safety Laboratory of Northwest A&F University (refer to "The Influence of HE Gene Receptor Binding Domain Variation on Bovine Coronavirus Infectivity. Northwest A&F University, 2024."); Primary antibodies: DDIT3 antibody (rabbit) was purchased from Shanghai Abmart, BCoV N protein antibody (mouse) was kindly provided by Professor Yin Xin of Harbin Veterinary Research Institute, and GAPDH (mouse) was purchased from Shanghai Abmart; Secondary antibodies: Horseradish peroxide (HRP) labeled goat anti-mouse IgG antibody was purchased from Shanghai Diyi Biotechnology Co., Ltd.; mRNA reverse transcription and quantification kits and qPCR kits were purchased from Beijing TransGen Biotech Co., Ltd.; PVDF membranes were purchased from Immobilon Biotechnology Co., Ltd.; ECL chromogenic solution was purchased from Beijing Dining Biotechnology Co., Ltd.; TurboFect was purchased from Thermo Fisher Scientific Co., Ltd.; all other routine reagents were of analytical grade.

[0068] The cultured HCT-8 cells were digested and counted beforehand, and then diluted to 2×10⁶ cells / mL with RPMI 1640 medium containing 10% FBS. 4 Add 1 mL of cell suspension to each well of the culture plate to a concentration of 1 cell / mL. Transfection can be performed when the cell density reaches 50%. Transfect the cells using TurboFect transfection reagent. DDIT3The gene siRNA, si-DDIT3, was used in the following cell groups: a negative control siRNA (si-NC), a virus-infected cell group (without siRNA transfection), and a normal cell group (without siRNA transfection and without virus infection). After transfection, the culture plates were incubated at 37°C in a 5% CO2 incubator for 48 h, and then infected with bovine coronavirus (MOI=10). After 48 h, the bovine coronavirus replication (mRNA and protein expression) levels were measured, and the viral TCID in the cell culture supernatant was also detected. 50 .

[0069] mRNA levels: Cells were collected 48 h after infection with bovine coronavirus. Total RNA was extracted from the collected cells using the TRIZOL precipitation method, and then the extracted total RNA was reverse transcribed into cDNA. The cDNA obtained from the reverse transcription was used as a template, and intracellular BCoV was detected using qPCR primers (Table 9). N Gene mRNA levels. Real-time quantitative PCR detection results are as follows: DDIT3 As shown in Figure A, cells transfected with si-DDIT3 can be seen to show the effect of BCoV infection 48 hours after infection with bovine coronavirus. N The expression level of the gene mRNA increased. This result indicates that the intracellular bovine coronavirus replication level increases with... Figure 5 Gene knockdown increases expression.

[0070] Protein expression levels: Cells were collected 48 h after infection with bovine coronavirus. Cells were lysed with RIPA lysis buffer containing a protease inhibitor, lysed on ice for 15 min, then loaded with loading buffer and boiled for 10 min. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. After blocking with 5% milk powder or 5% BSA for 2 h, the membrane was treated with primary and secondary antibodies before development. The expression levels of DDIT3 and BCoV N proteins in cells are shown below. DDIT3 As shown in B, knockdown is visible. Figure 5 The expression level of bovine coronavirus protein increased after gene sequencing.

[0071] TCID 50 Experiment: Cells transfected with si-NC, si-DDIT3, and untransfected siRNA were collected and their supernatants were collected 48 h after viral infection. One day in advance, HCT-8 cells in logarithmic growth phase were resuspended and their concentration adjusted to 8 × 10⁶ cells / cells. 4 Cells / mL were evenly inoculated into 96-well plates, with 8 replicates for each viral concentration gradient. The cell culture medium was discarded when the cell density reached approximately 80%. The cell culture supernatant collected after inoculation was serially diluted with RPMI 1640 medium to a dilution factor of 10-1. -1 Up to 10 -10Cell culture supernatant was added at a rate of 100 μL per well to the above-mentioned 96-well plate, while an equal volume of RPMI 1640 medium was added to the blank control wells. After waiting for virus adsorption for 2 h, the cells were replaced with RPMI 1640 medium containing 2% FBS. The CPE status was observed daily. Once the cell status in each well was stable and the number of diseased wells no longer increased, the viral titer (TCID) of the amplified bovine coronavirus was calculated according to the Reed-Muench method. 50 ), through TCID 50 The results of detecting viral titers in cell culture supernatants are as follows: As shown in C, compared to cells infected only with BCoV, knockdown... DDIT3 Re-infection with BCoV after gene mutation significantly increases the viral titer of BCoV in cell culture supernatant.

[0072] In summary, this invention has found through experiments that: (1) the expression level of DDIT3 in human host cells (e.g., HCT-8) increases after bovine coronavirus infection; and (2) RNA interference technology can be used to inhibit the expression of DDIT3 in human host cells (e.g., HCT-8). Figure 5 After gene expression, the downregulation of DDIT3 expression promotes the replication of bovine coronavirus, resulting in a significant increase in both the in vitro replication level and viral titer of bovine coronavirus; (3) while the introduction of overexpressing human into human host cells (e.g., HCT-8) DDIT3 Following plasmidization of the gene, upregulation of DDIT3 expression inhibited bovine coronavirus replication, significantly reducing both the in vitro replication level and viral titer. Therefore... DDIT3 This gene is a bovine coronavirus resistance gene and can be used for certain antiviral applications (such as through effective activation). DDIT3 DDIT3 DDIT3 It can inhibit the replication of bovine coronavirus by gene inhibition; at the same time, it can provide efficient viral amplification cells for research on the pathogenic mechanism of bovine coronavirus (such as the molecular mechanism of regulating viral replication) and the preparation of bovine coronavirus vaccines (inactivated bovine coronavirus vaccines, attenuated vaccines, etc.), thus providing new ideas for the prevention and control of bovine coronavirus and having broad application prospects.

Claims

1. A method for preparing high-titer bovine coronavirus, characterized in that: Includes the following steps: Knockdown of human cells DDIT3 Genes were used to obtain knockdown cells; the knockdown cells obtained by infection with bovine coronavirus were cultured, and then the cell culture supernatant was separated to obtain high-titer bovine coronavirus.

2. The method for preparing high-titer bovine coronavirus according to claim 1, characterized in that: The knockdown was performed using RNA interference technology.

3. The method for preparing high-titer bovine coronavirus according to claim 1, characterized in that: The knockdown specifically includes the following steps: transfecting human cells with siRNA with a sequence as shown in SEQ.ID.NO.

2.

4. The method for preparing high-titer bovine coronavirus according to claim 1, characterized in that: The human-derived cells are human ileocecal cancer cells.

5. A cell model that promotes bovine coronavirus infection, characterized in that: This model is based on the use of suppression DDIT3 It is made by treating human cells with gene expression agents or reagents.

6. The cell model for promoting bovine coronavirus infection according to claim 5, characterized in that: The formulation includes human... DDIT3 siRNA of genes.

7. A method for knocking down people DDIT3 Gene interference RNA, characterized by: The interfering RNA includes siRNA with a sequence as shown in SEQ.ID.NO.

2.

8. with DDIT3 Application of gene / DDIT3 protein-targeting activators in the preparation of antiviral drugs for bovine coronavirus.

9. The application according to claim 8, characterized in that: The drug includes a human DDIT3 protein overexpression vector.

10. A cell model for resisting bovine coronavirus infection, characterized in that: This model is based on using methods to promote DDIT3 It is made by treating human cells with gene expression agents or reagents.

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