Application of RAB8B gene in the prevention and treatment of swine Glasser's disease

Abstract

The present invention discloses RAB8B Application of genes in the prevention and treatment of pig Glaser's disease through targeted inhibition RAB8B Gene expression can effectively inhibit Haemophilus parasuis from adhering to and invading host cells and causing cytopathic effects, resisting cell death induced by Haemophilus parasuis infection, and thus preventing and treating swine Glasser's disease caused by Haemophilus parasuis infection. RAB8B The gene can serve as a potential target for the prevention and treatment of porcine Glasser's disease and has important clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and particularly relates to application of RAB8B gene as a target molecule in the prevention and treatment of porcine Glasser's disease. Background Art

[0002] Swine Glasser's disease, also known as polyfibrosing serositis and arthritis, is a bacterial infectious disease caused by Haemophilus parasuis (G. parasuis) and characterized by fibrinous polyserositis, arthritis, and meningitis. Clinically, it presents a syndrome characterized by fever, joint swelling, dyspnea, polycystic serositis, and high mortality, seriously endangering the health of weaned piglets and nursery pigs. There is currently no specific treatment for swine Glasser's disease. Although vaccines and antibiotics are available, their preventive and therapeutic effects are unsatisfactory. The fundamental reason for the current lack of effective prevention and control measures for swine Glasser's disease is that the pathogenic mechanism of G. parasuis is unclear and there is a lack of key and effective gene targets. Therefore, finding new therapeutic targets is particularly important for the development of host-targeted antibacterial drugs.

[0003] The RAB8B gene belongs to the RAB family, the largest subfamily of small GTP-binding proteins. RAB proteins are present in nearly all membrane-associated organelles of eukaryotic cells, maintaining the continuity of protein transport and the integrity of organelle membranes. Previous studies have shown that RAB8B can eliminate Mycobacterium tuberculosis through immune autophagy (Pilli M et al., 2012; Kalam Het al., 2017), suggesting that RAB8B participates in cellular pathways crucial for bacterial adhesion and invasion of the host. However, the relationship between RAB8B, a new antibacterial member, and swine Glasser's disease caused by Haemophilus parasuis has not been reported. Summary of the Invention

[0004] The present invention discloses a drug for the prevention and treatment of porcine Glasser's disease using the RAB8B gene as a target, wherein the drug is a substance that targets and inhibits the expression of the RAB8B gene. Specifically, the drug comprises an sgRNA or an expression vector thereof that targets and knocks down the RAB8B gene.

[0005] Further preferably, the sgRNA sequence for targeted knockout of the RAB8B gene is 5'-GGAAAAGCGGAATAGGAGGC-3'.

[0006] The present invention also discloses a drug comprising sgRNA and Cas protein for targeted knockdown of the RAB8B gene, and the drug can be used to prevent and treat porcine Glasser's disease.

[0007] The present invention also discloses the use of the RAB8B gene as a target in the preparation of gene-edited cells or animal models resistant to Haemophilus parasuis infection. Specifically, the method comprises: (1) using a lentiviral vector to package HEK293T cells to obtain CRISPR / Cas9 system lentiviral particles; (2) infecting a target cell line with the lentiviral particles, and screening the cells by flow cytometry to obtain a cell line with RAB8B gene knockdown.

[0008] Furthermore, the present invention found that the RAB8B gene is correlated with the adhesion and invasion of Haemophilus parasuis. In a specific embodiment of the present invention, knocking down the RAB8B gene in PK1 cells can reduce the ability of Haemophilus parasuis to infect PK1 cells and improve cell proliferation and survival.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention provides the use of the RAB8B gene in preventing and treating Glasser's disease in pigs. The present invention discovered that the expression level of the RAB8B gene is upregulated after Haemophilus parasuis infects host cells. Targeted knockdown of the RAB8B gene in host cells can effectively inhibit Haemophilus parasuis infection of host cells, thereby preventing and treating Glasser's disease caused by G. parasuis infection in pigs. Therefore, RAB8B can serve as a potential target for the prevention and treatment of Glasser's disease in pigs and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The mutation status of the RAB8B gene knockdown cell line detected by Sanger sequencing in Example 1. WT is a wild-type cell, KD is a RAB8B gene knockdown cell constructed using the CRISPR / Cas9 lentiviral strategy, PAM is the abbreviation for protospacer adjacent motif, and sgRNA is the abbreviation for small guide RNA.

[0012] Figure 2 The Western blotting technique was used to detect the RAB8B protein expression level in the RAB8B knockdown cell line in Example 1. β-actin is an internal reference gene, and kDa represents kilodaltons.

[0013] Figure 3 This is the EdU cell proliferation assay used in Example 2 to evaluate the effect of RAB8B knockdown on cell proliferation. WT represents wild-type cells, and KD represents RAB8B knockdown cells. Blue fluorescence indicates DAPI-stained nuclei, and red fluorescence indicates EdU-positive cells. ns indicates P > 0.05, indicating no significant difference.

[0014] Figure 4 To detect the mRNA expression level of RAB8B in PK1 cells at different time points after G. parasuis infection (MOI = 10) in Example 3 using fluorescence quantitative PCR technology; among them, GAPDH is the internal reference gene.

[0015] Figure 5 To detect the protein expression level of RAB8B in PK1 cells at different time points after G. parasuis infection (MOI = 10) in Example 3 using Western blotting technology. Among them, β-actin is the internal reference gene, and kDa represents kilodalton.

[0016] Figure 6 For the imaging results of the cell survival status of wild-type cells and RAB8B gene knockdown cells at 0 hour and 120 hours after G. parasuis infection (MOI = 10) in Example 4.

[0017] Figure 7 For the changes in cell viability of wild-type cells and RAB8B gene knockdown cells at different time points after G. parasuis infection (MOI = 10) in Example 4. , ,

[0018] , , Figure 7 , * , , Figure 9 , , Figure 8 , ,

[0022] , ** ,

[0021] ,

[0020] , ,

[0023] ,

[0019] Represents P < 0.01, extremely significant difference; * Represents 0.01 < P < 0.05, significant difference; ns represents P > 0.05, no significant difference.

[0018] Figure 8 To detect the amount of bacteria adhering to and invading cells of wild-type cells and RAB8B gene knockdown cells at different time points after G. parasuis infection (MOI = 10) in Example 5 using the plate counting method for bacteria spreading.

[0019] Figure 9 To detect the amount of bacteria invading cells of wild-type cells and RAB8B gene knockdown cells at different time points after G. parasuis infection (MOI = 10) in Example 5 using the plate counting method for bacteria spreading. Specific implementation mode

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

[0021] Example 1: Construction of RAB8B gene knockdown cell line using CRISPR / Cas9 technology

[0022] 1.1 Design of sgRNA and construction of expression vector

[0023] sgRNA was designed for the exon sequence of the RAB8B gene (Ensembl:ENSSSCG00000026571), with a sequence of 5'-GGAAAAGCGGAATAGGAGGC-3' and a PAM sequence of "AGG". The company synthesized the sgRNA primer pair, RAB8B-sgR-F: 5'-caccgGGAAAAGCGGAATAGGAGGC-3', RAB8B-sgR-R: 5'-aaacGCCTCCTATTCCGCTTTTCCc-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. Next, the annealed product was ligated to the lentiCRISPR v2 (addgene: #52961) vector linearized with BsmBI (NEB). After transformation, plating, and overnight culture, single colonies were picked the next day for Sanger sequencing. Positive clones were expanded and plasmids were extracted using an endotoxin removal kit. The resulting vector, which correctly expressed the sgRNA, was named "lentiCRISPR v2-RAB8B-KD."

[0024] 1.2 Preparation and identification of RAB8B gene knockdown cell lines

[0025] Lentiviral packaging was performed by transfecting HEK293T cells with pMD2.G:psPAX2:lentiCRISPR v2-RAB8B-KD at a mass ratio of 1:2:3. PK1 cells were then inoculated with lentivirus and positive cells were selected by flow cytometry for expansion. Genomic DNA was then extracted using the Tiangen DNA Extraction Kit (KG203). To target the sgRNA target genomic region, the amplification primer pair RAB8B-PCR-F: 5'-GCGAAGACGTATGATTATCTG-3', and RAB8B-PCR-R: 5'-TTCCTGTCTTTCAATCCGTTT-3' were designed using NCBI-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). PCR reactions were then performed using the amplification primer pair using the genomic DNA as a template. The PCR products were then analyzed by Sanger sequencing to confirm gene knockdown in 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 (RAB8B-KD) ( Figure 1), indicating that the RAB8B gene coding sequence had a gene mutation. Further, the total cell protein was extracted and the protein expression of RAB8B in RAB8B-KD cells was detected by Western blotting. Figure 2 As shown, compared with wild-type cells, RAB8B was hardly expressed in the knockdown cells, indicating that the RAB8B knockdown cell line was successfully constructed.

[0026] Example 2: Knockdown of RAB8B does not affect normal cell proliferation

[0027] The EdU cell proliferation experiment was used to evaluate the effect of RAB8B gene knockdown on the normal proliferation of PK1 cells. First, an equal volume of 2×EdU working solution (20μM) preheated at 37°C was taken and mixed with the original culture medium in the cell culture plate to make the final EdU concentration 1×, and incubated for 2 hours; then, pre-cooled 4% paraformaldehyde was used to fix the cells at room temperature for 15 minutes, and then the cells were rinsed and permeabilized with pre-cooled 0.3% TritonX-100 at room temperature for 10 minutes. After rinsing the cells again, Click reaction solution was added and incubated in the dark for 30 minutes at room temperature; finally, PBS was washed three times, DAPI staining solution was added, and incubated in the dark for 10 minutes. Observation under a fluorescence microscope showed that there was no significant difference in the proliferation of RAB8B gene knockdown cells compared with wild-type cells ( Figure 3 ).

[0028] Example 3: RAB8B 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 was extracted at 0 hpi, 12 hpi, 24 hpi, 48 hpi and 96 hpi according to the TRIZOL method, and the changes in the mRNA expression level of RAB8B were detected by fluorescence quantitative PCR. The quantitative detection primers of the RAB8B gene are: RAB8B-qPCR-F: 5'-CCTGCCTCCTATTCCGCTTT-3', RAB8B-qPCR-R: 5'-CCAGCATGATTCCCATGGCT-3'. The results showed that after infection with G. parasuis, the mRNA expression level of RAB8B in PK1 cells was significantly upregulated ( Figure 4 At the same time, total cell protein was extracted at 0 hpi, 48 hpi and 96 hpi, and the protein expression of RAB8B was detected by Western blotting. Figure 5 As shown in the figure, with the extension of G. parasuis infection time, the expression level of RAB8B protein in PK1 cells gradually increased.

[0030] Example 4: Knockdown of RAB8B can significantly inhibit host cell pathology caused by G. parasuis infection

[0031] To analyze the inhibitory effect of RAB8B knockdown on G. parasuis-induced cytopathic effect, wild-type cells and RAB8B knockdown cells were infected with G. parasuis at an MOI of 10. After incubation for 24 h, the growth medium was replaced. 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 using an inverted optical microscope. Figure 6 As shown, while wild-type cells showed obvious swelling and rupture, and cells died and fell off on a large scale, PK1 cells with RAB8B gene knockdown did not rupture, and a large number of cells survived.

[0032] At the same time, the cell viability at different time points (0, 6, 12, 24, 48, 72, and 120 hpi) of G. parasuis infection was detected using the CellTiter-Lumi Plus luminescence cell viability assay kit. Figure 7 As shown, there was no difference in the viability of RAB8B gene knockdown cells and wild-type cells in the G. parasuis uninfected group at different time points, further indicating that knocking down the RAB8B gene did not affect the normal growth of cells; the cell viability of RAB8B gene knockdown cells in the G. parasuis infected group was significantly higher than that of wild-type cells at 48, 72 and 120 hours after infection.

[0033] Therefore, knocking down the RAB8B gene can significantly inhibit the cytopathic effect caused by G. parasuis and significantly improve the survival rate of host cells. RAB8B knockdown cells have the ability to resist cell death induced by G. parasuis infection.

[0034] Example 5: Knockdown of RAB8B can significantly inhibit the adhesion and invasion of G. parasuis to host cells

[0035] Wild-type cells and RAB8B gene 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, 6, 12, 24 and 48 hpi), the cells were washed 3-5 times with warm PBS to remove non-specific adherent bacteria. After thorough digestion with 200 μL of trypsin, 800 μL of pre-cooled deionized water was added to completely lyse the cells. After the lysate was pipetted and mixed, 100 μL was taken at an appropriate dilution ratio and spread on TSA solid culture medium 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 number 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 shown in Figure 2. Figure 8 As shown, the number of bacteria on the surface and inside the RAB8B gene knockdown cells infected with G. parasuis for 12 and 24 hours was significantly lower than that in 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 then the number of bacteria invading PK1 cells was calculated using the plate plating method. Results: Compared with wild-type cells, the number of bacteria inside the RAB8B gene knockdown cells infected with G. parasuis for 12, 24 and 48 hours was significantly reduced ( Figure 9 ). This showed that knocking down the RAB8B gene could significantly inhibit the adhesion and invasion of G. parasuis into host cells.

[0036] In the above steps, any technology not described in detail or specifically specified is conventional technology in the prior art and can be performed according to conventional molecular biology and cell biology experimental conditions or the conditions recommended by the manufacturer's instructions.

[0037] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. Use of a RAB8B gene inhibitor in the preparation of a drug for preventing or treating porcine Glasser's disease, characterized in that: The inhibitor is a nucleotide or an expression vector thereof that targets and knocks down the RAB8B gene, and the sequence of the nucleotide is 5'-GGAAAAGCGGAATAGGAGGC-3'.

2. Application of a specific sgRNA targeting and knocking down the RAB8B gene in constructing a cell model or animal model against porcine Glasser's disease, characterized in that: The sequence of the sgRNA is 5'-GGAAAAGCGGAATAGGAGGC-3'.

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