Application of LYZ gene in promotion of African swine fever virus replication
By screening and constructing LYZ gene overexpression and knockout cell lines, the shortcomings in the study of ASFV replication mechanism were addressed, and the promoting or inhibiting effects of the LYZ gene in ASFV replication were realized, which can be applied to the development of African swine fever virus vaccines and drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LANZHOU VETERINARY RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES(LANZHOU BRANCH CENTER OF CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENTER)
- Filing Date
- 2026-01-19
- Publication Date
- 2026-04-21
AI Technical Summary
Current technologies do not adequately study the molecular regulatory mechanisms of ASFV replication, and there is a lack of targeted gene tools that can be used for research on viral replication mechanisms, vaccine development, and drug screening. The role of the LYZ gene in ASFV replication is unknown.
Differentially expressed genes were screened through transcriptomics analysis, and LYZ gene overexpression plasmids and knockout cell lines were constructed. Using LYZ gene/protein as targets, LYZ gene/protein expression inhibitors and overexpression vectors were developed for the preparation of drugs and vaccine enhancers.
Overexpression of the LYZ gene promotes ASFV replication, while knockout of the LYZ gene inhibits ASFV replication. The LYZ gene/protein can serve as an enhancer for African swine fever virus vaccines and a drug target for the prevention or treatment of African swine fever.
Smart Images
Figure CN121896341A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of molecular biology and virology, specifically involving LYZ New applications of genes, especially involving LYZ Application of genes in promoting African swine fever virus replication. Background Technology
[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by the African swine fever virus (ASFV). It has devastatingly impacted the global pig farming industry, and currently there are no effective vaccines or treatments. In-depth research into the ASFV replication mechanism and related host factors is crucial for developing prevention and control technologies.
[0003] In the current technology, the molecular regulatory mechanism of ASFV replication is not well understood, the number of host genes involved in viral replication has been limited, and there is a lack of targeted gene tools that can be used for research on viral replication mechanisms, vaccine development, and drug screening. LYZ The gene (lysozyme gene) is known to play a role in immune defense, but there are no reports on its association with ASFV replication. Whether it participates in and how it regulates ASFV replication remains unknown, and the relevant research gaps urgently need to be filled.
[0004] Viruses need to invade host cells and replicate, assemble, and release within them to complete their life cycle. On the one hand, viruses utilize the host cell's resources and energy to aid their replication; on the other hand, host cells can also defend against viral invasion through their own defense mechanisms. Therefore, in-depth research into the regulatory mechanisms between ASFV replication and host factors is of great significance for elucidating the pathogenic mechanisms of viral infection and for disease prevention and control. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention uses transcriptomics analysis to screen for differentially expressed genes in ASFV-susceptible cells (BMDM and PAM cells) and non-susceptible cells (iPAM cells). Subsequently, the top 18 genes were selected, expression plasmids were constructed, and transfected into 293T cells and iPAM cells for inoculation experiments. The results showed that overexpression of these genes... LYZ The gene promotes ASFV replication. Knockout LYZ The gene has an inhibitory effect on ASFV replication. Specifically, it includes the following: In a first aspect, the present invention provides a LYZ The application of gene / protein targets in the preparation or screening of drugs for the prevention or treatment of African swine fever virus infection; said drug is... LYZ Targeting genes / proteins to inhibit or silence them. LYZ Gene / protein expression.
[0006] Secondly, the present invention provides LYZ Application of gene / protein expression inhibitors in the preparation of drugs for the prevention or treatment of African swine fever virus infection.
[0007] Preferably, the LYZ Gene / protein expression inhibitors include targeted knockout LYZ sgRNA of genes / proteins.
[0008] Preferably, the sgRNA is selected from LYZ-sgRNA1 and LYZ-sgRNA2; The forward primer for LYZ-sgRNA1 is CACCGctcgcaccgatcatagacct, and the reaction primer is AAACaggtctatgatcggtgcgagC. The forward primer for LYZ-sgRNA2 is CACCGagggttgtcagagatccac, and the reaction primer is AAACtgtggatctctgacaaccctC.
[0009] Thirdly, the present invention provides LYZ Application of gene / protein knockout cell lines in breeding for resistance to African swine fever virus.
[0010] Preferably, the LYZ The method for constructing gene / protein knockout cell lines includes the following steps: (1) Preparation of specific targets LYZ sgRNA of genes / proteins; (2) Insert the double-stranded fragment of the sgRNA oligonucleotide prepared in step (1) into the multiple cloning site of the lentiviral vector expressing Cas9, and transfect cells to obtain a recombinant lentivirus that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence. (3) Transduce the host cells with the recombinant lentivirus prepared in step (2), pick single cells, inoculate and culture them to obtain LYZ Gene / protein knockout cell lines.
[0011] Fourthly, the present invention provides LYZ The application of gene / protein targets in the preparation or screening of African swine fever virus or African swine fever virus vaccine production enhancers; the drug is used as... LYZ Targeting genes / proteins to promote LYZ Gene / protein expression.
[0012] Fifthly, the present invention provides LYZ Gene / protein or expression LYZApplication of gene / protein vectors in the preparation of African swine fever virus or African swine fever virus vaccine production enhancers.
[0013] Sixthly, the present invention provides LYZ Application of gene / protein overexpression cell lines in the preparation of African swine fever virus or African swine fever virus vaccine production cell lines.
[0014] Preferably, the LYZ The method for constructing gene / protein overexpression cell lines includes the following steps: (1) Preparation LYZ Overexpression plasmids; (2) Constructing the structure described in step (1) LYZ Recombinant lentiviruses overexpressing plasmids; (3) Transduce the host cells with the recombinant lentivirus prepared in step (2), pick single cells, inoculate and culture them to obtain LYZ Overexpression cell lines.
[0015] The beneficial effects of this invention are: this invention first discovers overexpression through screening. LYZ The gene promotes the replication of ASFV. LYZ Genes / proteins or their overexpression vectors can be used as performance enhancers in the production of African swine fever virus vaccines; secondly, knockout... LYZ The gene can suppress ASFV replication. LYZ Genes / proteins can be used as targets to screen for drugs that inhibit the replication of African swine fever virus, for the prevention or treatment of African swine fever. Attached Figure Description
[0016] Figure 1 LYZ Gene overexpression plasmid pPB-EF1α-LYZ-Flag.
[0017] Figure 2 Results of LYZ gene protein expression verification.
[0018] Figure 3 Western blotting was used to detect the expression of LYZ protein.
[0019] Figure 4 The results of absolute quantitative PCR detection of ASFV p72 content in 293T cells overexpressing LYZ.
[0020] Figure 5 ASFV TCID in 293T cells overexpressing LYZ 50 Test results.
[0021] Figure 6Results of ASFV p72 content detection in iPAM cells overexpressing LYZ.
[0022] Figure 7 ASFV TCID in iPAM cells overexpressing LYZ 50 Test results.
[0023] Figure 8 Results of ASFV p72 content detection in LYZ knockout cells.
[0024] Figure 9 ASFV TCID in LYZ knockout cells 50 Test results. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the various embodiments of this invention will be described in detail below with reference to the accompanying drawings. However, those skilled in the art will understand that many technical details have been provided in the embodiments of this invention to facilitate a better understanding of this application. However, the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.
[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.
[0027] Example 1 LYZ Construction of gene overexpression cells (1) Construction LYZ Gene overexpression vector Searching for pigs on NCBI LYZ Download the gene sequence (Gene ID: 100157211). LYZ CDS sequence of the gene. mRNA was extracted from PAM cells and reverse transcribed into cDNA. Targets were designed using SnapGene. LYZ The upstream and downstream primers for the gene sequence were designed as follows: the upstream primer sequence is ttcaggtgtcgtgaAGCTAGCgccaccATGAAGACTCTCCTCGTTCTT (SEQ ID NO.1), and the downstream primer sequence is GTACATTCGGGGTTGCAAACTGAtggactataaggaccacgacggag (SEQ ID NO.2). The designed primer sequences were sent to General Biotech for artificial synthesis.
[0028] The PCR reaction system is shown in Table 1.
[0029] Table 1 PCR reaction system
[0030] The PCR reaction procedure is shown in Table 2.
[0031] Table 2 PCR reaction system
[0032] The PCR amplification products were subjected to agarose gel electrophoresis, and the target band was cut and recovered from the gel. The recovered product was then ligated with the Nhe I-digested pX459 vector backbone using a 2xG method. The ligation product was transformed into competent cells to obtain... LYZ Gene overexpression plasmid pPB-EF1α-LYZ-Flag (e.g.) Figure 1 (As shown).
[0033] (2) Pig LYZ Gene and protein expression verification 293T cells were seeded into 12-well plates. When the cell density reached 90% and the cell condition was good with 60-80% confluence, the cells were transfected using jetPRIME transfection reagent (jetPRIME® in vitro DNA & siRNA transfection reagent PROTOCOL, PT-114-15) according to the jetPRIME instructions. 2 μg of pPB-EF1α-LYZ-Flag plasmid was transfected into the 293T cells. 24 h after transfection, the EV group was treated with the empty pPB-EF1α-Flag vector, with the remaining steps the same as the experimental group. 48 h after transfection, 150 μL of lysis buffer was added to each well to extract cell proteins, and Western blotting was performed to detect LYZ protein expression. Results are as follows: Figure 2 As shown, the LYZ protein is approximately 16 kDa, and the protein size was as expected according to Western blotting.
[0034] iPAM cells were digested with trypsin. The amount of cells from one 10 cm cell culture dish was sufficient for electroporation into six wells of a 12-well plate. 2 μg of pPB-EF1α-LYZ-Flag plasmid was added to 200 μl of electroporation buffer and allowed to stand for 15 min. The corresponding amount of cells was then added and mixed thoroughly by pipetting. The mixture was transferred to an electroporation cuvette, and electroporation was performed using the Y-001 program on the electroporator. The EV group received the empty pPB-EF1α-Flag vector, and the remaining steps were the same as the experimental group. After electroporation and 48 h of recovery culture, 150 μl of lysis buffer was added to each well to extract cell proteins. Western blotting was then used to detect LYZ protein expression. Results are as follows: Figure 3As shown, LYZ protein expression was detected in both 293T-LYZ-Flag cells and iPAM-LYZ-Flag cells.
[0035] Example 2 LYZ Construction of knockout cell lines pX459 is a CRISPR / Cas9 system plasmid that can express Cas9 protein and transcribe sgRNA for gene knockout. LYZ-sgRNA1 and LYZ-sgRNA2 were cloned into this plasmid. Specific steps: The forward and reaction primers for LYZ-sgRNA1 were synthesized artificially. The forward primer was LYZ-sgRNA1-F: CACCGctcgcaccgatcatagacct (SEQ ID NO. 3), and the reaction primer was LYZ-sgRNA1-R: AAACaggtctatgatcggtgcgagC (SEQ ID NO. 4). The forward and reaction primers for LYZ-sgRNA2 were also synthesized artificially. The forward primer was LYZ-sgRNA2-F: CACCGagggttgtcagagatccaca (SEQ ID NO. 5), and the reaction primer was LYZ-sgRNA2-R: AAACtgtggatctctgacaaccctC (SEQ ID NO. 6). The above forward and reaction primers were annealed to form double-stranded fragments. The annealing system consisted of 5 μL of 10 × Annealing Buffer and 22.5 μM of forward primer (10 μM). μL of reverse primer (10 μM) and 22.5 μL of anti-reverse primer were mixed; annealing program: 95℃ for 5 min, then stored at 4℃; the above double-stranded fragment was ligated to the pX459 vector backbone digested with BbsI at T4, and the ligation product was transformed into competent cells to obtain... LYZGene targeting plasmids pX459-LYZ-gRNA1 and pX459-LYZ-gRNA2 were used. When wild-type WSL cells were in good condition and confluenced to 60-80%, iPAM cells were digested with trypsin. The amount of cells from one 10 cm cell culture dish was sufficient for electroporation into 6 wells of a 12-well plate. 2 μg of pPB-EF1α-LYZ-Flag plasmid was added to 200 μL of electroporation buffer and allowed to stand for 15 min. The corresponding amount of cells was then added and mixed thoroughly by pipetting. The mixture was transferred to an electroporation cuvette and electroporated using the Y-001 program on an electroporator. Control cells (WT) were treated with an equal volume of electroporation buffer but without the vector; the remaining steps were the same. 24 h after transfection, the cells were passaged and puromycin (2 μg / mL) was added to the culture medium. Transfected positive cells were screened out. 2-3 days after drug screening, the cells were cultured and expanded in 1640 medium containing 20% FBS. Since no single-clone screening was performed, the WSL-LYZ-KO mixed cell line was obtained in this experiment.
[0036] Example 3: Detection of the effect of LYZ protein overexpression on ASFV replication After infecting LYZ-overexpressing 293T cells and control cells prepared in Example 1 with ASFV, the intracellular ASFV p72 content was detected. The same number of iPAM-LYZ-Flag cells and control cells were infected with ASFV. After 24 h of infection, cells and supernatant were collected, and the cells and supernatant were repeatedly frozen and thawed three times at -80℃ and room temperature. The cells and supernatant were then collected again. The cells were centrifuged at 8000 rpm for 2 min, and cell debris was discarded, leaving the supernatant. The ASFV p72 content in the samples was detected using absolute quantitative PCR. The results are shown below. Figure 4 As shown, ASFV replication was significantly enhanced in LYZ-overexpressing cells compared to control cells.
[0037] After ASFV was used to infect LYZ-overexpressing 293T cells constructed in Example 1 and control cells, intracellular ASFV TCID was reduced. 50 Detection. The same number of iPAM-LYZ-Flag cells and control cells were infected with ASFV. 24 h after infection, cells and supernatant were collected, and the cells and supernatant were repeatedly frozen and thawed three times at -80℃ and room temperature. The cells and supernatant were then centrifuged at 8000 rpm for 2 min, discarding cell debris and retaining the supernatant. The obtained virus samples were then subjected to 10... -1 -10 -8Virus samples were diluted 100 μL per well and seeded into BMDM cells in 96-well cell culture plates at different dilutions. Five wells were inoculated with each dilution. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 96 h, and cytopathic effects were observed and recorded every 12 h. The TCID of the virus was calculated using the Reed-Muench method. 50 The result is as follows Figure 5 As shown, viral replication was significantly enhanced in LYZ-overexpressing 293T cells compared to control cells.
[0038] Overexpression of LYZ protein in iPAM, detection of intracellular ASFV p72 levels and intracellular ASFV TCID 50 The detection method was consistent with that used in 293T cells. The results were as follows: Figure 6 and Figure 7 As shown, viral replication was significantly enhanced in LYZ-overexpressing iPAM cells compared to control cells.
[0039] The above results indicate that overexpression LYZ The gene promotes the replication of ASFV. LYZ Genes / proteins or their overexpression vectors can be used as enhancement agents in the production of African swine fever virus vaccines.
[0040] Example 4: Detection of the effect of LYZ protein knockout on ASFV replication In the WSL-KO-MIX hybrid knockout cells constructed in Example 2, intracellular ASFV p72 levels and ASFV TCID were detected. 50 The detection method was the same as that used for detecting ASFV p72 content and ASFV TCID in 293T-LYZ-Flag cells. 50 Testing.
[0041] The results are as follows Figure 8 and Figure 9 As shown, compared to control cells, the ASFV p72 content in knockout cells was significantly reduced ( Figure 8 As shown), viral replication in knockout cells was significantly inhibited ( Figure 9 (As shown).
[0042] The above results indicate that knockout LYZ The gene can suppress ASFV replication. LYZ Genes / proteins can be used as targets to screen for drugs that inhibit the replication of African swine fever virus, for the prevention or treatment of African swine fever.
Claims
1. LYZ The application of gene / protein targets in the preparation or screening of drugs for the prevention or treatment of African swine fever virus infection; said drug is... LYZ Targeting genes / proteins to inhibit or silence them. LYZ Gene / protein expression.
2. LYZ Application of gene / protein expression inhibitors in the preparation of drugs for the prevention or treatment of African swine fever virus infection.
3. The application as described in claim 2, characterized in that, The LYZ Gene / protein expression inhibitors include targeted knockout LYZ sgRNA of genes / proteins.
4. The application as described in claim 3, characterized in that, The sgRNA is selected from LYZ-sgRNA1 and LYZ-sgRNA2; The forward primer for LYZ-sgRNA1 is CACCGctcgcaccgatcatagacct, and the reaction primer is AAACaggtctatgatcggtgcgagC. The forward primer for LYZ-sgRNA2 is CACCGagggttgtcagagatccac, and the reaction primer is AAACtgtggatctctgacaaccctC.
5. LYZ Application of gene / protein knockout cell lines in breeding for resistance to African swine fever virus.
6. The application as described in claim 5, characterized in that, The LYZ The method for constructing gene / protein knockout cell lines includes the following steps: (1) Preparation of specific targets LYZ sgRNA of genes / proteins; (2) Insert the double-stranded fragment of the sgRNA oligonucleotide prepared in step (1) into the multiple cloning site of the lentiviral vector expressing Cas9, and transfect cells to obtain a recombinant lentivirus that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence. (3) Transduce the host cells with the recombinant lentivirus prepared in step (2), pick single cells, inoculate and culture them to obtain LYZ Gene / protein knockout cell lines.
7. LYZ The application of gene / protein targets in the preparation or screening of African swine fever virus or African swine fever virus vaccine production enhancers; the drug is used as... LYZ Targeting genes / proteins to promote LYZ Gene / protein expression.
8. LYZ Gene / protein or expression LYZ Application of gene / protein vectors in the preparation of African swine fever virus or African swine fever virus vaccine production enhancers.
9. LYZ Application of gene / protein overexpression cell lines in the preparation of African swine fever virus or African swine fever virus vaccine production cell lines.
10. The application as described in claim 8, characterized in that, The LYZ The method for constructing gene / protein overexpression cell lines includes the following steps: (1) Preparation LYZ Overexpression plasmids; (2) Constructing the structure described in step (1) LYZ Recombinant lentiviruses overexpressing plasmids; (3) Transduce the host cells with the recombinant lentivirus prepared in step (2), pick single cells, inoculate and culture them to obtain LYZ Overexpression cell lines.