Application of RHOA gene in prevention and treatment of swine Graisseria disease

Abstract

The invention discloses an application of an RHOA gene in prevention and treatment of swine Graisseria, which can effectively inhibit adhesion and invasion of haemophilus parasuis to host cells and reduce cytopathy by targeted inhibition of RHOA gene expression. The method is helpful for resisting cell death induced by haemophilus parasuis infection, so as to prevent the occurrence of the porcine Graisseria disease. Therefore, the RHOA gene can be used as an important target spot for treating the swine Graisseria disease, and has an important clinical application prospect.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to the application of RHOA gene as a target molecule in the prevention and treatment of swine Glasser's disease. Background Art

[0002] Swine Glasser's disease, also known as polyfibrinous serositis and arthritis, is a bacterial infectious disease caused by Haemophilus parasuis (G. parasuis). Clinically, it manifests as fever, joint swelling, difficulty breathing, polyserositis and high mortality, posing a serious threat to the health of weaned piglets and nursery pigs. Although there are vaccines and antibiotics, the prevention and treatment of swine Glasser's disease is not ideal. At present, the pathogenic mechanism of the disease is still unclear, and there is a lack of key and effective therapeutic targets. Therefore, finding new gene targets is particularly urgent and important for the development of host-targeted antibacterial drugs.

[0003] The RHOA gene (Ras Homolog Family Member A) encodes the small GTPase RhoA, which belongs to the Rho family. RhoA can cycle between the active GTP-bound state and the inactive GDP-bound state, acting as a molecular switch in the cell to regulate a variety of signal transduction pathways. RhoA regulates vesicle transport, cell adhesion, fusion, and movement by activating signal cascades, and participates in the host infection process of various viruses, such as herpes virus, adenovirus, dengue virus, poxvirus, pseudorabies virus, etc.

[0004] Although RHOA plays an important role in a variety of viral infections, its role in swine Glasser's disease caused by Haemophilus parasuis is still unclear. Further research on the function of RHOA in this specific bacterial disease is expected to provide new ideas for future treatment strategies. Summary of the invention

[0005] The present invention discloses a drug for preventing and treating swine Glasser's disease using the RHOA gene as a target, wherein the drug is a substance that targets and inhibits the expression of the RHOA gene. Specifically, the drug includes an sgRNA or an expression vector thereof that targets and knocks down the RHOA gene.

[0006] Further preferably, the sgRNA sequence for targeted knockout of the RHOA gene is 5'-GAGGTATATGTACCTACCGT-3'.

[0007] The present invention also discloses a drug, comprising sgRNA for targeted knockdown of the RHOA gene, and the drug can be used to prevent and treat porcine Glasser's disease.

[0008] The present invention also discloses the use of the RHOA gene as a target in the preparation of gene-edited cells or animal models resistant to Haemophilus parasuis bacterial infection. Specifically, it includes: (1) using a lentiviral vector to package HEK293T cells to obtain CRISPR / Cas9 system lentiviral particles; (2) infecting the target cell line with the lentiviral particles, and screening by flow cytometry to obtain a cell line with RHOA gene knockdown.

[0009] Furthermore, the present invention found that the RHOA gene is correlated with the adhesion and invasion of Haemophilus parasuis. In a specific embodiment of the present invention, when the RHOA gene is knocked down, the ability of Haemophilus parasuis to infect PK1 cells can be reduced, and the cell proliferation ability and survival rate can be improved.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention provides an application of the RHOA gene in preventing and treating swine Glasser's disease. The present invention finds that the expression level of the RHOA gene is upregulated after Haemophilus parasuis infects host cells, and targeted knockdown of the RHOA gene expression can effectively inhibit Haemophilus parasuis from infecting host cells, thereby preventing and treating swine Glasser's disease caused by G. parasuis infection. Therefore, the RHOA gene can be used as a potential target for the prevention and treatment of swine Glasser's disease, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The mutation of the RHOA knockdown cell line detected by Sanger sequencing in Example 1. Wherein, WT is a wild-type cell, RHOA-KD is a RHOA gene knockdown cell constructed by CRISPR / Cas9 lentiviral strategy, PAM is the abbreviation of protospacer adjacent motif, and sgRNA is the abbreviation of small guide RNA.

[0013] Figure 2 The Western blotting technique was used to detect the RHOA protein expression level of the RHOA knockdown cell line in Example 1. β-actin is an internal reference gene, and kDa represents kilodaltons.

[0014] Figure 3 This is the effect of RHOA gene knockdown on cell proliferation evaluated by EdU cell proliferation assay in Example 2. Blue fluorescence is DAPI-stained cell nuclei, and red fluorescence is EdU-stained positive cells. ns means P>0.05, no significant difference.

[0015] Figure 4To detect the mRNA expression level of RHOA in PK1 cells at different time points during G. parasuis infection (MOI = 10) in Example 3 by fluorescence quantitative PCR; where GAPDH is the internal reference gene. ** Indicates P < 0.01, highly significant difference; * Indicates 0.01 < P < 0.05, significant difference.

[0016] Figure 5 It is the imaging result diagram of the cell survival status of wild - type cells and RHOA - knockdown cells at 0 hour and 120 hours during G. parasuis infection (MOI = 10) in Example 4.

[0017] Figure 6 It is the cell proliferation situation of wild - type cells and RHOA - knockdown cells at 48 hours during G. parasuis infection (MOI = 10) in Example 4. The blue fluorescence is the nucleus stained by DAPI, and the red fluorescence is the EdU - stained positive cells. ** Indicates P < 0.01, highly significant difference.

[0018] Figure 7 It is the change in cell viability of wild - type cells and RHOA - knockdown cells at 72 hours and 120 hours during G. parasuis infection (MOI = 10) in Example 4. ** Indicates P < 0.01, highly significant difference.

[0019] Figure 8 To detect the amount of bacteria adhering to and invading cells (upper figure) and the amount of bacteria invading cells (lower figure) at different time points of wild - type cells and RHOA - knockdown cells during G. parasuis infection (MOI = 10) by plate - coating bacteria counting method in Example 5. Detailed implementation manners

[0020] The technical solutions of the present invention are described in detail through the following specific examples.

[0021] Example 1: Construction of RHOA gene - knockdown cell line using CRISPR / Cas9 technology

[0022] 1.1 sgRNA design and expression vector construction

[0023] sgRNA was designed for the exon sequence of the RHOA gene (Ensembl: ENSSCG00000022080), and its sequence was 5'-GAGGTATATGTACCTACCGT-3', and the PAM sequence was "CCG". The company was sent to synthesize the sgRNA primer pair, RHOA-sgR-F: 5'-caccgACGGTAGGTACATATACCTC-3', RHOA-sgR-R: 5'-aaacGAGGTATATGTACCTACCGTc-3'. Subsequently, 5 μL of the synthesized sgRNA primers F and R (10 pmol) were taken and annealed in a PCR instrument: 95°C, 10 min; 65°C, 60 min. Then, the annealed product was connected to the lentiCRISPR v2 (addgene: #52961) vector linearized by BsmBI (NEB). Further, transformation, plating, and overnight culture were performed, and a single colony was picked for sanger sequencing identification the next day. Positive clones were selected for expansion and culture, and the plasmid was extracted using an endotoxin removal kit. The vector with correct expression of sgRNA was named "lentiCRISPR v2-RHOA-KD".

[0024] 1.2 Preparation and identification of RHOA gene knockdown cell lines

[0025] HEK293T cells were transfected with pMD2.G:psPAX2:lentiCRISPR v2-RHOA-KD at a mass ratio of 1:2:3 for lentivirus packaging. Subsequently, PK1 cells were inoculated with lentivirus, and strong positive cells were selected by flow sorting for expansion culture. Then, the genomic DNA of the cells was extracted according to the method of Tiangen DNA extraction kit (KG203). Targeting the sgRNA target genomic region, the amplification primer pair was designed using NCBI-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) as RHOA-PCR-F: 5'-GGCCTGAGACTATTATGGCTT-3', RHOA-PCR-R: 5'-CACATATTCTAAGGCTTCCCAATG-3'. Then, genomic DNA was used as a template, and the amplification primer pair was used for PCR reaction, and the PCR product was sequenced by Sanger to identify the gene knockdown of the cells. The results showed that compared with wild-type cells (WT), there were non-single peaks in the sgRNA target region in the genome of knockdown cells (RHOA-KD) ( Figure 1 ), indicating that the RHOA gene coding sequence had a gene mutation. Further, the total cell protein was extracted and the protein expression of RHOA in RHOA-KD cells was detected by Western blotting. Figure 2 As shown, compared with wild-type cells, RHOA was hardly expressed in the gene knockdown cells, indicating that the RHOA knockdown cell line was successfully constructed.

[0026] Example 2: Knockdown of RHOA does not affect normal cell proliferation

[0027] The EdU cell proliferation experiment was used to evaluate the effect of RHOA gene knockdown on the normal proliferation of PK1 cells. First, take an equal volume of 2×EdU working solution (20μM) preheated at 37°C and mix it with the original culture solution in the cell culture plate to make the final EdU concentration 1×, and incubate for 2h; then fix with precooled 4% paraformaldehyde at room temperature for 15min, then rinse the cells, permeabilize with precooled 0.3% TritonX-100 at room temperature for 10min, rinse the cells again, add Click reaction solution and incubate at room temperature in the dark for 30min; finally, wash with PBS 3 times, add DAPI dye solution, and incubate in the dark for 10min. Observation under a fluorescence microscope found that there was no significant difference in the proliferation of RHOA gene knockdown cells and wild-type cells ( Figure 3 ).

[0028] Example 3: RHOA gene expression level is upregulated after G. parasuis infects PK1 cells

[0029] At an MOI of 10, G. parasuis (serotype 5 SH0165 strain, the same below) was inoculated to infect PK1 cells. Total cell RNA at 0 hpi, 12 hpi and 48 hpi was extracted using the TRIZOL method, and the changes in the mRNA expression level of RHOA were detected by fluorescence quantitative PCR. The quantitative detection primers of the RHOA gene are: RHOA-qPCR-F: 5'-CGTTGGTCTTGCAGCACATT-3', RHOA-qPCR-R: 5'-TACCTCCGGGAATTGGTCCT-3'. The results showed that after infection with G. parasuis, the mRNA expression level of RHOA in PK1 cells was significantly upregulated ( Figure 4 ).

[0030] Example 4: Knockdown of RHOA can significantly inhibit host cell lesions caused by G. parasuis infection

[0031] In order to analyze the inhibitory effect of RHOA knockdown on G. parasuis-induced cytopathic effect, wild-type cells and RHOA knockdown cells were infected with G. parasuis at an MOI of 10. The growth medium was replaced after 24 h of incubation. The growth medium was replaced every day until almost all wild-type cells in the infected group died (120 h). The cytopathic effect was observed and photographed under an optical inverted microscope. Figure 5As shown, while wild-type cells showed obvious swelling and rupture, and cells died and fell off on a large scale, PK1 cells with RHOA gene knockdown did not rupture, and a large number of cells survived.

[0032] 48 h after infection, the EdU cell proliferation assay was used to evaluate the effect of RHOA gene knockdown on the normal proliferation of PK1 cells (the operation was the same as in Example 2). Figure 6 As shown, 48 hours after infection, the proliferation capacity of RHOA knockdown cells was significantly higher than that of wild-type cells.

[0033] At the same time, the CellTiter-Lumi Plus luminescent cell viability detection kit was used to detect the cell viability at different time points (72 and 120 hpi) infected with G. parasuis, such as Figure 7 As shown, there was no difference in the viability of RHOA gene knockdown cells and wild-type cells in the G. parasuis uninfected group at different time points, further indicating that knocking down the RHOA gene did not affect the normal growth of cells; the cell viability of RHOA gene knockdown cells in the G. parasuis infected group was significantly higher than that of wild-type cells at 72 and 120 hours after infection.

[0034] Therefore, knocking down the RHOA gene can significantly inhibit the cytopathic effect caused by G. parasuis and significantly improve the survival rate of host cells. RHOA knockdown cells have the ability to resist cell death induced by G. parasuis infection.

[0035] Example 5: Knockdown of RHOA can significantly inhibit the adhesion and invasion of G. parasuis to host cells

[0036] Wild-type cells and RHOA knockdown cells were infected with G. parasuis at an MOI of 10. After incubation for 24 hours, the bacteria were removed and the growth medium was replaced. At different incubation time points (1, 3, 9, 12 and 24 hpi), the cells were washed 3-5 times with warm PBS to remove non-specific adherent bacteria. After adding 200 μL of trypsin for thorough digestion, 800 μL of pre-cooled deionized water was added to completely lyse the cells; after the lysate was blown and mixed, 100 μL was spread on TSA solid culture medium at an appropriate dilution ratio and incubated at 37°C for 36 hours; in addition, parallel samples were set up and the cell count was calculated using a hemocytometer; finally, the amount of bacteria adhering to and invading each cell was counted (for specific methods, refer to "Assessment of the Macrophage Scavenger Receptor CD163 in Mediating Glaesserella parasuis Infection of Host Cells" published in Veterinary Sciences in 2023). The results are as follows Figure 8 As shown, the number of bacteria on the surface and inside the RHOA gene knockdown cells infected with G. parasuis for 3, 9, 12 and 24 hours was significantly lower than that of the wild-type cell group. At the same time, parallel samples were set up. Before lysing the cells, G. parasuis adhered to the cell surface was removed by gentamicin and penicillin, and then the amount of bacteria invading PK1 cells was calculated by the plate plating method. Results: Compared with wild-type cells, the number of bacteria inside the RHOA gene knockdown cells infected with G. parasuis for 3, 9, 12 and 24 hours was significantly reduced. This shows that knocking down the RHOA gene can significantly inhibit the adhesion and invasion of G. parasuis to host cells.

[0037] In the above steps, the techniques not described in detail or specified are all conventional techniques in the prior art and can be performed according to conventional molecular biology and cell biology experimental conditions or according to the conditions recommended by the manufacturer's instructions.

[0038] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. RHOA Use of a gene inhibitor, characterized in that: Used for preparing medicine for preventing or treating swine Glasser's disease.

2. The use according to claim 1, characterized in that Said RHOA Gene inhibitors including targeted knockdown RHOA The nucleotide sequence of the gene or its expression vector.

3. A drug for the prevention or treatment of porcine Glaser's disease, characterized in that: Including targeted knockdown RHOA The nucleotide sequence of the gene or its expression vector.

4. The drug according to claim 4, characterized in that Including targeted knockdown RHOA The specific sgRNA of the gene, the sgRNA sequence is 5'-GAGGTATATGTACCTACCGT-3'.

5. A cell model or animal model for resistance to porcine Glaser's disease, characterized in that: In cells RHOA The gene was knocked down. RHOA Protein expression was inhibited.

Images