Application of TRAPPC2 gene in regulation and control of infection ability and intracellular survival of animal immune cells to Brucella

Abstract

The invention belongs to the technical field of genetic engineering, and relates to application of a TRAPPC2 gene in regulation and control of infection ability and intracellular survival of animal immune cells to brucella, and the nucleotide sequence of the TRAPPC2 gene is shown as SEQ ID NO: 1. After the TRAPPC2 gene in the mononuclear macrophage is knocked out, the infection ability of the Brucella to the mononuclear macrophage can be remarkably reduced, intracellular survival of the Brucella in immune cells is remarkably inhibited, the infection rate of the Brucella is remarkably reduced, and the Brucella can be effectively prevented from being infected. It is shown that the TRAPPC2 gene is a key gene for regulating and controlling the infection ability of animal immune cells to Brucella and intracellular survival. The TRAPPC2 gene disclosed by the invention can provide a new candidate molecular material for animal disease-resistant breeding work and related drug research and development; meanwhile, a new research direction and a new technical thought are provided for clinical treatment of brucellosis in the medical field.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving TRAPPC2 Application of genes in regulating the ability of animal immune cells to infect Brucella and their intracellular survival. Background Technology

[0002] Brucellosis is caused by Brucella bacteria ( Brucella Brucella (Brucella sp.) is a globally prevalent zoonotic Class B infectious disease. Its prevalence not only threatens public health but also causes significant economic losses to the livestock industry, hindering capacity expansion. The core mechanism of Brucella pathogenicity relies on its ability to efficiently infect host immune cells (especially innate immune cells such as monocytes / macrophages and dendritic cells) and its ability to evade immune clearance and establish stable replication within the cell. Therefore, reducing Brucella infection efficiency directly reduces the initial bacterial load, while weakening Brucella's intracellular survival limits its spread and proliferation in host tissues, thereby alleviating systemic inflammatory responses and toxic symptoms, ultimately affecting disease progression and severity.

[0003] In recent years, the global incidence of brucellosis has been increasing year by year, but its prevention and treatment face severe challenges: there are currently no specific drugs or highly effective and safe vaccines, and clinical treatment mainly relies on long-term combined use of antibiotics. This not only easily leads to the emergence of drug-resistant strains, but also has problems such as long treatment cycles and obvious side effects, putting dual pressure on public health security and the sustainable development of the livestock industry. Therefore, maintaining biosecurity and public health order, identifying key host genes that regulate Brucella infection and intracellular survival, and enhancing the resistance of farmed animal immune cells to Brucella infection through targeted regulation of these genes are of great significance for reducing antibiotic overuse, promoting the development of new vaccines and targeted drugs, improving livestock farming productivity, and building a comprehensive brucellosis prevention and control system. Based on this, identifying key host factors against Brucella and clarifying their regulatory mechanisms, and then developing targeted prevention and control technologies and products, are the core technical problems that urgently need to be solved in the field of brucellosis prevention and control. Summary of the Invention

[0004] The purpose of this invention is to provide TRAPPC2 The application of genes in regulating the ability of animal immune cells to infect Brucella and their intracellular survival, the aforementioned TRAPPC2 It can provide new candidate molecular materials for animal disease-resistant breeding and related drug development; at the same time, it also provides new research directions and technical ideas for the clinical treatment of brucellosis in the medical field.

[0005] This invention provides TRAPPC2 The application of genes in regulating the ability of animal immune cells to infect Brucella and their intracellular survival, the aforementioned TRAPPC2The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0006] As a preferred embodiment, the TRAPPC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:2.

[0007] As a preferred embodiment, the regulation includes inhibiting the immune cells of the animal. TRAPPC2 Gene expression or knockout of animal immune cells TRAPPC2 Gene ， It inhibits the ability of Brucella to infect animal immune cells, inhibits the intracellular survival of Brucella in animal immune cells, and reduces the Brucella infection rate.

[0008] The present invention also provides a negatively adjustable TRAPPC2 Biological materials that inhibit gene expression, including those that suppress gene expression. TRAPPC2 Gene expression or knockout TRAPPC2 Gene-based biological materials; The suppression method includes silencing; The TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0009] As a preferred solution, suppress the TRAPPC2 Gene expression or knockout TRAPPC2 Biological materials containing the gene include: TRAPPC2 Silencing vectors containing the gene silencing target, TRAPPC2 One or more of the following: a gene knockout target knockout vector, a transgenic cell line containing the silencing vector, a transgenic cell line containing the knockout vector, a recombinant microorganism containing the silencing vector, and a recombinant microorganism containing the knockout vector.

[0010] As a preferred embodiment, the basic framework of the knockout vector includes the lenti-cas9-blast vector and the lentiGuide-sg-eGFP vector.

[0011] As a preferred embodiment, when the knockout vector is a gene editing vector, it further includes sgRNA; the sgRNA includes sgRNA1 and sgRNA2; the nucleotide sequence of sgRNA1 is shown in SEQ ID NO:3, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO:4.

[0012] The present invention also provides applications of the aforementioned biomaterials, including at least one of the following: (1) Application in improving animal immune function; (2) Application in breeding animals with high immunity; (3) Application in animal disease-resistant breeding; (4) Application in constructing animal models of brucellosis resistance.

[0013] The present invention also provides a method for enhancing the anti-brucellosis effect of immune cells, comprising the following steps: knocking down target immune cells TRAPPC2 Gene expression levels or knockout in target immune cells TRAPPC2 Genes that produce immune cells that resist Brucella; The TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0014] The present invention also provides anti-brucellosis immune cells obtained using the method described above.

[0015] Beneficial effects: This invention provides TRAPPC2 The application of genes in regulating the ability of animal immune cells to infect Brucella and their intracellular survival, the aforementioned TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1. This invention knocks out [the gene] in monocytes / macrophages. TRAPPC2 Following gene administration, the ability of Brucella to infect mononuclear macrophages was significantly reduced, the intracellular survival of Brucella in immune cells was significantly inhibited, and the infection rate of Brucella was significantly reduced, indicating that the aforementioned TRAPPC2 Genes are key genes that regulate the ability of animal immune cells to infect Brucella and their intracellular survival. The present invention describes... TRAPPC2 Genes can provide new candidate molecular materials for animal disease-resistant breeding and related drug development; at the same time, they also provide new research directions and technical ideas for the clinical treatment of brucellosis in the medical field.

[0016] This invention, through phylogenetic trees and sequence similarity matrices of TRAPPC2 in different species, found that the amino acid sequence of TRAPPC2 has high similarity (>90%) among different species, indicating that TRAPPC2 has the potential to regulate Brucella invasion and intracellular survival across species. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a graph showing the optimal lethal concentration of BSD for Thrp-1 cells; where A represents the control group; and B represents the cell death rate at a BSD concentration of 20 μg / ml. Figure 2 These are validation images for Thrp-1-Cas9; where A is the PCR validation image; B is the qPCR validation image; and C is the WB validation image. P ≤0.05, P ≤0.01, P ≤0.001, P ≤0.0001; Figure 3 The graph shows the optimal lethal concentration of Tp-1Cas9 puro; where A is the control group; and B is the cell death rate at a puro concentration of 2.5 μg / ml. Figure 4 This is a graph showing the results of plasmid quality testing for the library. Figure 5 The results of the screening criteria are shown in the figure; where A represents the proportion of viable THP-1-Cas9 cells under different challenge conditions; and B represents the THP-1-Cas9 infection efficiency (proportion of RED+ cells) under different challenge conditions. Figure 6 The results of screening key host factors against Brucella in the library are shown in the figure; where A is the screening flowchart; B is the box plot of normalized sgRNA counts in the control group and experimental group before and after two repeated screenings; and C is the Venn diagram of differentially expressed genes in two positive screenings. Figure 7 This is a diagram showing the construction of the TRAPPC2 KO cell line; where A is a schematic diagram of the knockout; and B is a diagram showing the mRNA validation results. P ≤0.05, P ≤0.01, P ≤0.001, P ≤0.0001; Figure 8 for TRAPPC2 The effect of knockout on Brucella adhesion and invasion is shown in the figure; where A represents... TRAPPC2 Knockout B. Melitensis The effects of 16M adhesion and intrusion; B is TRAPPC2 Knockout B. Abortus The effects of 2308 adhesion and intrusion; P ≤0.05, P ≤0.01, P ≤0.001, P ≤0.0001; TC is the control group (Thp-1 Cas9 cells), 16M is... B. Melitensis 16M, S2308 are B. Abortus 2308; Figure 9 for TRAPPC2 The effect of knockout on Brucella intracellular survival is shown in the figure; where A represents... TRAPPC2 Knockout B. Melitensis The effect of 16M on intracellular survival; B is TRAPPC2 Knockout B. Abortus The impact on the intracellular survival of 2308 cells; P ≤0.05, P ≤0.01, P ≤0.001, P ≤0.0001; TC is the control group (Thp-1 Cas9 cells), 16M is... B. Melitensis 16M, S2308 are B. Abortus 2308; Figure 10 for TRAPPC2 The effect of knockout on Brucella infection rate is shown in the figure; where A is... TRAPPC2 Knockout B. Melitensis The effect of 16M infection rate; B is TRAPPC2 Knockout B. Abortus The impact of 2308 infection rate; P ≤0.05, P ≤0.01, P ≤0.001, P ≤0.0001; CON represents the control group, Thrp-1 Cas9 cells; Figure 11 Figure 1 shows the phylogenetic tree and amino acid sequence alignment results among different species of TRAPPC2; where A is the phylogenetic tree and B is the amino acid sequence alignment. Detailed Implementation

[0019] This invention provides TRAPPC2 Application of genes in regulating the ability of animal immune cells to infect Brucella and their intracellular survival; as a specific implementation method, the... TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1: 5'--3'.

[0020] As one specific implementation method, the TRAPPC2The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:2: MSSWKQDRSGLRSTELNVLEYQPLCAVRSHILKTMSGSFYFVIVGHHDNPVFEMEFLPAGKAESKDDHRHLNQFIAHAALDLVDENMWLSNNMYLKTVDKFNEWFVSAFVTAGHMRFIMLHDIRQEDGIKNFFTDVYDLYIKFSMNPFYEPNSPIRSSAFDRKVQFLGKKHLLS. This invention, through phylogenetic trees and sequence similarity matrices of TRAPPC2 in different species, found that the amino acid sequence of TRAPPC2 has high similarity (>90%) among different species, indicating that TRAPPC2 has the potential to regulate Brucella invasion and intracellular survival across species.

[0021] As one specific implementation, the regulation includes inhibiting animal immune cells. TRAPPC2 Gene expression or knockout of animal immune cells TRAPPC2 Gene ， It inhibits the ability of Brucella to infect animal immune cells, inhibits the intracellular survival of Brucella in animal immune cells, and reduces the Brucella infection rate.

[0022] The present invention also provides a negatively adjustable TRAPPC2 Biological materials that inhibit gene expression, including those that suppress gene expression. TRAPPC2 Gene expression or knockout TRAPPC2 Biological materials for genes; the method of inhibition includes silencing; the TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0023] As one specific implementation method, suppressing the TRAPPC2 Gene expression or knockout TRAPPC2 Biological materials containing the gene include: TRAPPC2 Silencing vectors containing the gene silencing target, TRAPPC2 One or more of the following: a gene knockout target knockout vector, a transgenic cell line containing the silencing vector, a transgenic cell line containing the knockout vector, a recombinant microorganism containing the silencing vector, and a recombinant microorganism containing the knockout vector.

[0024] In one specific embodiment, the basic framework of the knockout vector includes the lenti-cas9-blast vector and the lentiGuide-sg-eGFP vector. In another specific embodiment of the present invention... TRAPPC2The knockout vectors for the gene knockout target sites are Lenti-TRAPPC2-sg1-eGFP knockout vector and Lenti-TRAPPC2-sg2-eGFP knockout vector, obtained by ligating sgRNA1 and sgRNA2 into the lentiGuide-sg-eGFP vector, respectively; the nucleotide sequence of sgRNA1 is shown in SEQ ID NO:3: 5'-GTGTCGGCATTTGTCACTGCG-3'; the nucleotide sequence of sgRNA2 is shown in SEQ ID NO:4: 5'-TGTCGGCATTTGTCACTGC-3'.

[0025] In one specific embodiment, the recombinant microorganism containing the knockout vector includes DH5α containing the Lenti-TRAPPC2-sg1-eGFP knockout vector and DH5α containing the Lenti-TRAPPC2-sg2-eGFP knockout vector. In another specific embodiment, the transgenic cell line containing the knockout vector includes a cell line that knocks out a specific gene in THP-1. TRAPPC2 The transgenic cell line TRAPPC2 KO.

[0026] As one specific implementation method, the overexpression described TRAPPC2 Biological materials containing overexpressed genes include: TRAPPC2 One or more of the following: a recombinant expression vector of a gene, a transgenic cell line containing the recombinant expression vector, and a recombinant microorganism containing the recombinant expression vector.

[0027] The present invention also provides applications of the aforementioned biomaterials, including at least one of the following: (1) Application in improving animal immune function; (2) Application in breeding animals with high immunity; (3) Application in animal disease-resistant breeding; (4) Application in constructing animal models of brucellosis resistance.

[0028] The present invention also provides a method for enhancing the anti-brucellosis effect of immune cells, comprising the following steps: knocking down target immune cells TRAPPC2 Gene expression levels or knockout in target immune cells TRAPPC2 Genes, to obtain immune cells resistant to Brucella; the aforementioned TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1. As a specific embodiment, this invention describes the process of transforming Lenti-TRAPPC2-sg1-eGFP knockout vector and Lenti-TRAPPC2-sg2-eGFP knockout vector into Thrp-1 cells to obtain TRAPPC2 KO immune cells resistant to Brucella.

[0029] The present invention also provides anti-brucellosis immune cells obtained using the method described above.

[0030] The experiments of this invention show that knocking out Thp-1 cells TRAPPC2 After gene modification, Brucella bacteria will be significantly reduced. B. Melitensis 16M B. Abortus 2308 significantly reduced Brucella's ability to invade host cells in the early (1 hour) period. B. Melitensis 16M B. Abortus The intracellular survival level of 2308 was significantly reduced, and the infection rate of Brucella erythrocyte 16M-RED and S2308-RED was also significantly decreased 4 h. These results indicate that... TRAPPC2 Knockout of Brucella significantly reduces Brucella invasion of mononuclear macrophages and significantly reduces Brucella intracellular survival.

[0031] To further illustrate the present invention, the following description, in conjunction with embodiments, explains the features provided by the present invention. TRAPPC2 The application of genes in regulating the ability of animal immune cells to infect Brucella and their intracellular survival is described in detail, but should not be construed as limiting the scope of protection of this invention.

[0032] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0033] The experimental materials used in this invention are shown in Table 1, and the primers used in this invention are shown in Table 2.

[0034] Table 1 Test Materials Table 2 Primer sequence list

[0035] Example 1: Construction of a Thrp-1 knockout library 1. First, construct THP-1 cells that stably express the Cas9 protein. To screen for cell lines that stably express Cas9, it is necessary to determine the minimum concentration of an antibiotic (blastcin) that can kill untransfected THP-1 cells: (1) Thrp-1 cells were seeded and cultured overnight in 96-well plates (3×10⁻⁶ cells). 5 , 10% FBS 1640 complete culture medium).

[0036] (2) Set concentration gradient: Add blast fungicide to the complete culture medium to make the final concentrations 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, and 50 μg / ml. Make 6 parallel wells for each concentration.

[0037] (3) Replace the culture medium with freshly prepared blast fungicide every 3 days.

[0038] (4) Starting from day 7, observe the cell viability under a microscope. The lowest concentration that can kill cells is the working concentration for stable transfected cell selection, observed until all cells in the wells are observed to be dead. The final concentration is 20 μg / ml. Figure 1 ).

[0039] (5) 293T cell plating (0.7×10⁻⁶) 6 Transfection was initiated using 2% FBS antibiotic-free DMEM medium. Transfection began when cell confluence reached 80%, replacing the medium with fresh 2% FBS antibiotic-free DMEM before transfection. The transfection system was prepared according to the JetPrime transfection reagent instructions. The DNA composition included: packaging plasmid psPAX2, envelope plasmid pMD2.G, and target plasmid lenti-cas9-blast. Cell culture supernatant was collected 48 hours after transfection. Cell debris was removed using a 0.45 μm filter, and the collected lentiviral packaging solution was stored at -80°C for later use.

[0040] (6) Thrp-1 cells were administered at a dose of 1.2 × 10⁻⁶. 6 Cells were plated. The culture system for infected cells consisted of 1 ml of lentivirus packaging solution + 1 ml of 2% FBS antibiotic-free 1640 medium, while the culture system for uninfected cells consisted of 2 ml of 2% FBS antibiotic-free 1640 medium. Cells were continuously infected for 48 hours before being replaced with complete medium.

[0041] (7) After 72 hours, the lentivirus-infected and uninfected groups were replaced with 1640 complete culture medium containing the minimum lethal concentration of blast fungicide. Every 4 days, the culture medium was replaced with freshly prepared medium containing 20 μg / ml blast fungicide.

[0042] (8) Once all cells in the uninfected group have died, the drug screening is complete. Flow cytometry is used to sort surviving single cells into 96-well plates. Single cell growth is observed, and cells are labeled (for one week). Supplementation with fluid is maintained. Once single cell clusters have grown, they are transferred to 48-well plates for expanded culture, and 1×10⁶ cells are collected. 6 Thp-1-Cas9 cells and wild-type 1×10 6 Thp-1 cells were used, and DNA, RNA, and protein were extracted separately (the reagents for DNA, RNA, and protein extraction are shown in Table 1).

[0043] (9) Design Cas9-specific PCR primers (Table 2), use Thrp-1-Cas9 and Thrp-1 DNA as templates and follow the instructions of TAKARA Primerstar for PCR, prepare 1% agarose gel, run the gel at 120V for 30min, irradiate the gel and observe the target band. The target band was only observed at 129bp in Thrp-1-Cas9 cells. Figure 2 (A) DNA level detection indicates that Thrp-1-cas9 was successfully constructed.

[0044] (10) Design Cas9-specific qPCR primers (Table 2), use Thrp-1-Cas9 and Thrp-1 RNA as templates, and perform qPCR according to the instructions of Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) and ChamQ UniversalSYBR qPCR Master Mix. Calculate the results for each sample. cas9 The relative expression levels of the genes showed that Thrp-1 was almost not expressed at the mRNA level. cas9 Genes, compared to THP-1 cells and THP-1-Cas9 cells cas9 Significantly improved at the gene level ( Figure 2 The mRNA level detection (B) indicates that the Thip-1-cas9 was successfully constructed.

[0045] (11) Prepare PAGE gel (1.5 mm, 10-well comb) using Biotides 7.5% SDS-PAGE one-step rapid gel preparation kit. Add THP-1-cas9 and THP-1 proteins to Total Gold 6× Protein Loading Buffer in proportion. Prepare protein samples in a 95℃ metal bath for 10 min. Load 10 μl of THP-1-cas9 and THP-1 protein samples respectively. Run the gel at constant voltage of 80V for 30 min, 120V for 90 min, and transfer the membrane at 250mA for 2 h. Block with 5% skim milk powder in PBST for 2 h. Incubate with Flag primary antibody (primary antibody: PBST = 1:10000) at room temperature for 2 h. Wash with PBST 3 times for 10 min each time. Incubate with mouse secondary antibody (secondary antibody: PBST = 1:5000) at room temperature for 45 min. Wash with PBST 3 times for 10 min each time. Develop and photograph the membrane. The results showed that only the Thrp-1-Cas9 group had a flag band observed at 177 kDa. Figure 2 The protein level test results indicate that the Thip-1-cas9 was successfully constructed.

[0046] 2. The method for preparing the Thrp-1 knockout library cells is as follows: (1) Screening for the lowest lethal concentration of Thp-1 Cas9 puromycin: The method was the same as that for Thp-1 puromycin, with a lethal concentration of 2.5 μg / ml. Figure 3 ).

[0047] (2) The library plasmid (Gecko v2 human library A) was sent to Beijing Tangtang Tianxia Biotechnology Co., Ltd. for NGS quality testing, requiring sgRNA coverage > 99%. Figure 4 ).

[0048] (3) The packaging plasmid psPAX2, the envelope plasmid pMD2.G, the packaging library plasmid Gecko v2 humanlibrary A, the transfection and drug screening methods of the lentiviral packaging library plasmid are the same as the method of constructing THP-1 Cas9 cells.

[0049] (4) Once it is confirmed that all cells in the uninfected group have died, the drug screening is complete. The surviving cells are collected by flow cytometry and cultured to obtain whole-genome knockout library cells that can be used for subsequent functional screening.

[0050] Example 2: Library screening for key host factors against Brucella 1. To reduce false positives in screening results, it is necessary to optimize the screening challenge conditions, as follows: (1) The Tp-1-cas9 cells that stably express cas9 protein obtained above were divided into 1×10⁻⁶ cells. 6 Seeds were inoculated per well in a 6-well plate, with each well containing 1 ml of 2% serum-free antibiotic-free 1640 medium.

[0051] (2) 16M-RED was used to infect Thp-1-cas9 cells for 4h, 5h and 6h at MOI (bacterial number / cell number ratio) of 7500 and 10000 respectively.

[0052] (3) Add 2 μl of 50 μg / ml gentamicin to each well to kill extracellular bacteria for 1 hour.

[0053] (4) After killing the extracellular bacteria, mix the cells by pipetting and place them in a 1.5ml centrifuge tube. Centrifuge at 1100rpm for 8min and discard the supernatant. Gently resuspend and wash three times with PBS.

[0054] (5) Add 100 μl of Zombie UV™ Fixable Viability Kit to the cell pellet from step (4) to fix the cell pellet and resuspend it. Incubate at room temperature in the dark for 30 min.

[0055] (6) Centrifuge at 1100 rpm for 8 min, remove the supernatant, add 300 μl of 4% PFA fixative and fix for more than 30 min, filter, and perform flow cytometry analysis.

[0056] (7) The infection rate is the percentage of RED+ viable cells, and the proportion of viable cells is analyzed simultaneously. For example... Figure 5 As shown, the proportion of live cells ( Figure 5 (A) At MOI=7500, the proportion of viable cells did not decrease significantly with prolonged infection time (4h→5h→6h) (all ≥90%); at MOI=10000, the proportion of viable cells decreased significantly with prolonged infection time (e.g., it may be below 80% at 6h). Conclusion: The challenge concentration of MOI=7500 is less cytotoxic to THP-1-Cas9 cells, can maintain high host cell viability, and avoids the impact of massive cell death on subsequent screening results.

[0057] Infection efficiency ( Figure 5 (B) At MOI=7500 and 4h of infection, the proportion of RED+ cells was ≥90%. With prolonged infection time (5h, 6h), the infection efficiency did not significantly increase. At MOI=10000, although the infection efficiency approached 90% in a short time (4h), combined with the viable cell ratio, it was found that cytotoxicity increased. Conclusion: The combination of MOI=7500 and 4h of infection can maintain high cell viability (index A ≥90%) while achieving efficient Brucella infection of Thp-1-Cas9 cells (index B ≥90%), providing optimal conditions of "high cell viability + high infection rate" for subsequent library screening.

[0058] When 16M-RED cells were infected with MOI 7500 for 4 hours, the infection rate and viable cell ratio of THP-1-Cas9 cells could reach over 90%, which is the optimal infection condition.

[0059] 2. After determining the screening criteria, use 16M-RED to challenge bacteria and screen for host genes against Brucella. The procedure is as follows: (1) 2×10 8 2 × 10Thp-1 knockout library cells 6 Seeds were inoculated per well in 6-well plates with 4 ml of 2% serum-free 1640 medium.

[0060] (2) The experimental group infected the Thrp-1 knockout library cells with 16M-RED at MOI 7500. After 4 hours, 2 μl of 50 μg / ml gentamicin was added to each well to kill extracellular bacteria for 1 hour.

[0061] (3) Mix the cells by pipetting and centrifuging at 1100 rpm for 8 min, remove the supernatant, and gently resuspend and wash twice with PBS.

[0062] (4) Centrifuge at 1100 rpm for 8 min, remove the supernatant, add 3 ml of 4% PFA fixative and fix for at least 30 min, filter, and separate 2×10 using a flow cytometer. 7RED cells.

[0063] (5) Centrifuge at 2000×g for 20 min, remove supernatant, flash freeze in liquid nitrogen, and store. Collect 2×10⁻⁶ g of the control group. 7 Cells from the pre-infection Thrp-1 knockout library were centrifuged at 1100 rpm for 8 min, flash-frozen in liquid nitrogen, and then stored. Both cell groups were sent to Beijing Tangtang Tianxia Biomedical Information Technology Co., Ltd. for high-throughput sequencing and differential analysis. The screening was repeated twice, and the host factor obtained from both screenings was selected as candidate genes. Figure 6 A in the diagram represents the screening process. Figure 6 Box B shows the normalized sgRNA levels in the control and experimental groups before and after two repeated screenings. Figure 6 In the middle C, there is a Venn diagram of differentially expressed genes from two positive screenings. In total, this invention screened out 35 candidate host genes.

[0064] Example 3: Functional Verification of TRAPPC2 1. This invention mainly targets candidate host genes. TRAPPC2 Functional verification was performed using CRISPR / Cas9 knockout. TRAPPC2 After gene administration, the ability of mononuclear macrophages to resist Brucella was significantly improved. The operation method is as follows: (1) Two pairs of sgRNAs were designed using the CRISPOR (tefor.net) website. The nucleotide sequence of sgRNA1 is shown in SEQ ID NO:3: 5'-GTGTCGGCATTTGTCACTGCG-3'; the nucleotide sequence of sgRNA2 is shown in SEQ ID NO:4: 5'-TGTCGGCATTTGTCACTGC-3'. The sequences were synthesized by Beijing Xianghong Biotechnology Co., Ltd.

[0065] (2) Linearization of LentiGuide-sg-eGFP vector: 6µg vector, 10µl CutSmart Buffer, 3µl BsmBI, and ddH2O were added to 100 μl, and the mixture was incubated overnight in a water bath at 55℃. The sample was then identified and recovered from the gel.

[0066] (3) 5 µl of each of TRAPPC2-sg1-F, TRAPPC2-sg1-R, TRAPPC2-sg2-F, and TRAPPC2-sg2-R were annealed. The annealing program was as follows: 95℃ for 5 min, 95-85℃ for 2℃ / cyc for 0.7s for 5 cyc, 85-25℃ for 0.1℃ / cyc for 0.7s for 600 cyc, and 4℃ for ∞.

[0067] (4) Vector ligation: Ligation system: 5×Ligase Buffer 2μl, T4 ligase 1μl, linearized LentiGuide-sg-eGFP vector 50ng, annealed sgRNA 6μl, ddH2O to make up to 10μl, 25℃ for 10min, (3) annealed TRAPPC2-sg1 and TRAPPC2-sg2 are ligated to the vector respectively.

[0068] (5) Transformation plating: Thaw competent cells (DH5α) on ice, add 10µl of recombinant ligation product to 100µl of competent cells, incubate on ice for 30min, heat shock at 42℃ for 90s, immediately cool on ice for 5min, add 500µl of TSB culture medium and shake at 200rpm for 1h at 37℃. Collect bacteria at 7000rpm for 3min, discard the supernatant, resuspend in the remaining culture medium, and plating onto ampicillin-resistant plates. Incubate at 37℃ for 16h. On day 2, pick transformed single clones and shake to test for successful ligation. Successfully ligated plasmids are named Lenti-TRAPPC2-sg1-eGFP and Lenti-TRAPPC2-sg2-eGFP, respectively.

[0069] (6) Expand the culture of the successfully ligated monoclonal bacterial culture and extract the plasmid using the Tiangen endotoxin-free plasmid extraction kit.

[0070] (7) The packaging plasmid psPAX2 and the envelope plasmid pMD2.G were used to package the successfully constructed Lenti-TRAPPC2-sg1-eGFP and Lenti-TRAPPC2-sg2-eGFP, respectively. The resulting viral solutions were mixed in a 1:1 ratio and transfected into THP-1 Cas9 cells. The transfection method was the same as that used for constructing THP-1 Cas9 cells.

[0071] (8) After lentivirus infection for 48 hours, cells were collected, and GFP+ monoclonal cells were sorted by flow cytometry and transferred to 96-well plates. The growth status of single cells was observed and labeled (for one week). In addition of fluid was provided. After the single cell clusters grew, they were transferred to 48 (24)-well plates for expanded culture and mRNA extraction for detection. The detection method was the same as that used for the Thp-1 Cas9 cells. TRAPPC2 Specific qPCR primers are shown in Table 2, such as... Figure 7 As shown in B, TRAPPC2 KO TRAPPC2 The relative expression level was significantly reduced, and TRAPPC2 KO was successfully constructed.

[0072] 2. The TRAPPC2 KO monoclonal knockout cell line was constructed using the CRISPR / Cas9 gene editing system to verify its adhesion, invasion, and intracellular survival functions. The procedure is as follows: (1) Control group: Thrp-1 Cas9 and TRAPPC2 KO cells were administered at 3×10⁻⁶. 5 Seeds were inoculated per well in a 24-well plate with 1 ml of 2% serum-free 1640 medium.

[0073] (2) B. Melitensis 16M B. Abortus 2308 cells were infected with control group Thrp-1 Cas9 and TRAPPC2 KO cells at an MOI of 100.

[0074] (3) One hour after infection, 1 µl of 50 µg / ml gentamicin was added to each well for sterilization. Cell samples were collected at 1 hour after infection (unsterilized group, adhesion), 1 hour after sterilization (invasion), 4 hours, 12 hours, 24 hours and 48 hours after infection.

[0075] (4) After centrifugation at 1100 rpm for 5 min, discard the supernatant, add 500 µl of 0.1% Triton to resuspend and pipette, and after 1 min at room temperature, perform serial dilution and spot count to detect the invasion level and intracellular survival of Brucella. Repeat three times for each group and each time point.

[0076] 3. Using Thrp-1 as a template, the TRAPPC2 KO cell line was constructed using the CRISPR / Cas9 gene editing system for infection rate functional validation. The procedure is as follows: (1) Control group: Thrp-1 Cas9 and TRAPPC2 KO cells were administered at a dose of 1×10⁻⁶. 6 Seeds were inoculated per well in 6-well plates with 2 ml of 2% serum-free antibiotic-free 1640 medium.

[0077] (2) 16M-RED and S2308-RED were used to infect control group Thrp-1 Cas9 and TRAPPC2 KO cells with an MOI of 1000, respectively. (3) 4 hours after infection, add 2µl of 50µg / ml gentamicin to each well for sterilization for 1 hour, mix by pipetting and collect cells, centrifuge at 1100rpm for 8 minutes, remove the supernatant, and gently resuspend and wash twice with PBS.

[0078] (4) Fix with 300µl of 4% PFA fixative for more than 30 minutes, filter, and analyze the proportion of RED+ cells using a flow cytometer. Repeat three times for each group and each time point.

[0079] The results showed that in Thrp-1 cells, knocking out TRAPPC2 Does not affect Brucella B. Melitensis 16M B. Abortus 2308's ability to adhere to host cells ( Figure 8 China A Figure 8(Tables B, 3, and 4) significantly reduce Brucella bacteria. B. Melitensis 16M B. Abortus 2308's ability to invade host cells in the early stage (1 hour) Figure 8 China A Figure 8 (Tables B, 3, and 4) significantly reduced Brucella bacteria. B. Melitensis 16M B. Abortus 2308 intracellular survival level ( Figure 9 China A Figure 9 (See Tables B, 5, and 6). Flow cytometry results indicate that knockout... TRAPPC2 It significantly reduces the infection rate of Brucella erythrocyte 16M-RED and S2308-RED bacteria 4h. Figure 10 China A Figure 10 (B) The above results indicate TRAPPC2 Knockout of Brucella significantly reduces Brucella invasion of mononuclear macrophages and significantly reduces Brucella intracellular survival.

[0080] Table 3 TRAPPC2 Knockout B. Melitensis The effects of 16M adhesion and intrusion

[0081] Table 4 TRAPPC2 Knockout B. Abortus The effects of 2308 adhesion and intrusion

[0082] Table 5 TRAPPC2 Knockout B. Melitensis Effects of 16M intracellular survival

[0083] Table 6 TRAPPC2 Knockout B. Abortus Effects on 2308 intracellular survival

[0084] based on TRAPPC2 Genes regulate the function of Brucella invasion and intracellular survival, and have a significant impact on... TRAPPC2To perform cross-species sequence alignment and phylogenetic tree construction, visit the Uniprot website (https: / / www.uniprot.org / ), enter the target protein TRAPPC2 and click search. From the search results, select fully annotated, non-redundant TRAPPC2 protein sequences from representative species including humans, chickens, mice, cattle, pigs, sheep, and goats. Then, click Tools, select the Align tool, confirm the selected sequences are correct, and submit the alignment task. After alignment, click the corresponding blue link to view the sequence alignment results and conservation information. On the results page, view the phylogenetic tree of TRAPPC2 in different species. Figure 11 (A) and sequence similarity matrix ( Figure 11 (B) This invention found that the amino acid sequence of TRAPPC2 has high similarity (>90%) among different species, indicating that TRAPPC2 has the potential to regulate Brucella invasion and intracellular survival across species.

[0085] This shows that knocking out monocytes and macrophages... TRAPPC2 Following gene administration, the ability of Brucella to infect mononuclear macrophages was significantly reduced, the intracellular survival of Brucella in immune cells was significantly inhibited, and the infection rate of Brucella was significantly reduced, indicating that the aforementioned TRAPPC2 Genes are key genes that regulate the ability of animal immune cells to infect Brucella and their intracellular survival. The present invention describes... TRAPPC2 Genes can provide new candidate molecular materials for animal disease-resistant breeding and related drug development; at the same time, they also provide new research directions and technical ideas for the clinical treatment of brucellosis in the medical field.

[0086] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. TRAPPC2 The application of genes in regulating the ability of animal immune cells to infect Brucella and their intracellular survival is characterized by, The TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The TRAPPC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:

2.

3. The application according to claim 1 or 2, characterized in that, The regulation includes inhibiting animal immune cells. TRAPPC2 Gene expression or knockout of animal immune cells TRAPPC2 Gene ， It inhibits the ability of Brucella to infect animal immune cells, inhibits the intracellular survival of Brucella in animal immune cells, and reduces the Brucella infection rate.

4. A negative regulation TRAPPC2 Biomaterials with high gene expression levels are characterized by, Including suppressing the TRAPPC2 Gene expression or knockout TRAPPC2 Gene-based biological materials; The suppression method includes silencing; The TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

5. The biomaterial according to claim 4, characterized in that, Suppress the TRAPPC2 Gene expression or knockout TRAPPC2 Biological materials containing the gene include: TRAPPC2 Silencing vectors containing the gene silencing target, TRAPPC2 One or more of the following: a gene knockout target knockout vector, a transgenic cell line containing the silencing vector, a transgenic cell line containing the knockout vector, a recombinant microorganism containing the silencing vector, and a recombinant microorganism containing the knockout vector.

6. The biomaterial according to claim 5, characterized in that, The basic framework of the knockout vector includes the lenti-cas9-blast vector and the lentiGuide-sg-eGFP vector.

7. The biomaterial according to claim 4, characterized in that, When the knockout vector is a gene editing vector, it also includes sgRNA; the sgRNA includes sgRNA1 and sgRNA2; the nucleotide sequence of sgRNA1 is shown in SEQ ID NO:3, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO:

4.

8. The application of the biomaterial according to any one of claims 4 to 7, characterized in that, Includes at least one of the following: (1) Application in improving animal immune function; (2) Application in breeding animals with high immunity; (3) Application in animal disease-resistant breeding; (4) Application in constructing animal models of brucellosis resistance.

9. A method for enhancing the resistance of immune cells to Brucella, characterized in that, Includes the following steps: Knock down target immune cells TRAPPC2 Gene expression levels or knockout in target immune cells TRAPPC2 Genes that produce immune cells that resist Brucella; The TRAPPC2 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

10. Antibrucell immune cells obtained using the method of claim 9.

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