Application of TAB2 protein in rabies treatment target

Abstract

The application of TAB2 protein in rabies treatment target spot belongs to the field of biological medicine technology. In order to study the influence of host protein on RABV replication, further study the pathogenic molecular mechanism of RABV, and find new rabies treatment target and drug, the application uses fluorescence quantification, Western Blotting and other methods to study the influence of host protein TAB2 on RABV replication, and finds that TAB2 gene transient knockdown or stable knockdown can inhibit the replication of rabies virus, and further finds that TAB2 protein can be used as a new target for rabies treatment. In addition, the application also finds the structure domain of TAB2 and RABV M interaction and the TAB2 protein promotes RABV replication by activating TAK1-p38 / MAPK signal pathway, and the p38 inhibitor Gossypetin and the TAK1 inhibitor Takinib can be used as potential drugs for treating RABV infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of TAB2 protein as a therapeutic target for rabies. Background Technology

[0002] Rabies is an acute, highly fatal zoonotic infectious disease caused by the rabies virus (RABV), which can infect almost all warm-blooded animals. The disease is characterized by rapid onset and high mortality. Infected animals or patients often exhibit clinical features such as restlessness, hydrophobia, photophobia, and difficulty swallowing. Once clinical symptoms appear, the mortality rate can reach 100%. According to data from the World Health Organization (WHO), rabies ravages more than 150 countries worldwide, causing approximately 59,000 deaths annually, with about 95% of cases occurring in Asia and Africa. Therefore, studying the impact of host factors on RABV replication from the perspective of virus-host interactions, deeply analyzing its regulatory mechanisms, and screening for novel therapeutic targets and drugs are of great significance for the clinical treatment and comprehensive prevention and control of rabies. This also provides theoretical support for the global goal of eliminating canine-transmitted human rabies by 2030.

[0003] The RABV genome is a non-segmented, single-stranded, negative-sense RNA, approximately 12 kb in length, encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L), from the 3' to the 5' end. Among these, the RABV protein, a multifunctional protein, not only influences viral particle morphology, pathogenicity, assembly, budding, and release, but also participates in regulating viral transcription and replication, playing a crucial role in RABV replication.

[0004] TAB2 (TAK1-binding protein 2) is a binding protein to transforming growth factor β-activated kinase 1 (TAK1), with a full length of 693 amino acids and a relative molecular mass of approximately 77 kDa. TAB2 commonly binds to TAB1, TAB3, and TAK1, forming the TAK1-TAB1-TAB2 / 3 complex, and exerts its biological functions by regulating the activity of TAK1 protein. TAB2 participates in the regulation of multiple signaling pathways, including NF-κB and MAPK. However, whether TAB2 affects RABV replication has not yet been reported.

[0005] Rabies is a significant zoonotic infectious disease that threatens global public health and impacts world economic development. Its pathogenic mechanism is currently unclear, and there are no effective treatments. Therefore, studying the impact of host proteins on RABV replication from the perspective of host protein-virus interactions is of great importance for further research into the molecular mechanisms of RABV pathogenesis and for identifying new therapeutic targets and drugs. Summary of the Invention

[0006] To investigate the impact of host proteins on RABV replication, further study the molecular mechanisms of RABV pathogenesis, and identify new therapeutic targets and drugs for rabies, this invention focuses on the interaction between host proteins and the virus. It utilizes methods such as quantitative fluorescence, Western blotting, and direct immunofluorescence to study the influence of the host protein TAB2 on RABV replication, providing a theoretical basis for further research on the molecular mechanisms of RABV pathogenesis and the identification of new therapeutic targets and drugs.

[0007] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention proposes the following technical solution:

[0008] The first object of the present invention is to provide the use of a substance capable of inhibiting the expression of murine TAB2 protein in the preparation of a product for the treatment of rabies, characterized in that the murine TAB2 protein has the NCBI accession number NP_001346463.1.

[0009] In one embodiment of the present invention, the substance capable of inhibiting the expression of murine TAB2 protein is used to treat rabies by inhibiting the MAPK signaling pathway.

[0010] In one embodiment of the present invention, the rabies is caused by RABV CVS11 strain or RABV SRV9 strain.

[0011] A second object of the present invention is to provide the use of a substance capable of inhibiting the expression of a murine TAB2 protein in the preparation of a product for inhibiting the replication of rabies virus, wherein the murine TAB2 protein has the NCBI accession number NP_001346463.1.

[0012] In one embodiment of the present invention, the substance capable of inhibiting the expression of murine TAB2 protein inhibits rabies virus replication by inhibiting the MAPK signaling pathway.

[0013] In one embodiment of the present invention, the rabies virus is either RABV CVS11 strain or RABV SRV9 strain.

[0014] In one embodiment of the present invention, the substance capable of inhibiting the expression of mouse TAB2 protein is a substance that knocks out or knocks down the expression of TAB2 protein.

[0015] In one embodiment of the present invention, the substance used to knock down TAB2 protein expression is TAB2 siRNA.

[0016] In one embodiment of the present invention, the TAB2 siRNA is siRNA1 with a nucleotide sequence as shown in SEQ ID NO.9, or siRNA2 with a nucleotide sequence as shown in SEQ ID NO.10, or siRNA3 with a nucleotide sequence as shown in SEQ ID NO.11, or a mixture of siRNA1, siRNA2 and siRNA3.

[0017] In one embodiment of the present invention, the substance for knocking out TAB2 protein expression is a gene editing tool for knocking out TAB2 protein expression.

[0018] The beneficial effects of this invention are:

[0019] This invention, from the perspective of host protein-virus interaction, utilizes quantitative fluorescence, Western blotting, and direct immunofluorescence to study the effect of the host protein TAB2 on RABV replication. The results show that transient knockdown or stable knockout of the TAB2 gene can inhibit rabies virus replication, thus revealing that the TAB2 protein could serve as a novel therapeutic target for rabies. Furthermore, this invention also discovers that the TAB2 protein and RABV M protein co-localize within cells, and the interaction domain between TAB2 and RABV M is RABVM. 77-202 TAB2 51-574 and TAB2 575-693 And TAB2 575-693 The TAB2 protein is a key domain that promotes RABV replication in N2a cells. During RABV infection, the TAB2 protein promotes phosphorylation of the MAPK signaling pathway and has a significant impact on the p38 / MAPK and JNK / MAPK signaling pathways. It was found that the TAB2 protein promotes RABV replication through the TAK1-p38 / MAPK signaling pathway. The p38 inhibitor Gossypetin and the TAK1 inhibitor Takinib may be potential drugs for the treatment of RABV infection, which further provides a theoretical basis for the treatment of rabies and the development of drugs for the treatment of rabies. Attached Figure Description

[0020] Figure 1 The figure shows the effect of RABV infection on host TAB2 expression; where, Figure 1Figure A in the graph shows the results of RT-qPCR detection of the effect of RABV infection on the transcriptional level of the host factor TAB2 mRNA. Figure 1 In the figure, B represents the results of Western blotting to detect the effect of RABV infection on the expression level of the host factor TAB2 protein. Figure 1 C in the text is a pair Figure 1 The result of performing grayscale analysis on B in the image;

[0021] Figure 2 A schematic diagram of the pcDNA3.1-musTAB2 recombinant plasmid;

[0022] Figure 3 The image shows the results of Western blotting analysis to detect the overexpression efficiency of TAB2 protein; in which, Figure 3 Figure A in the graph represents the results of Western blotting detection of TAB2 protein overexpression efficiency. Figure 3 B in the text is a pair Figure 3 The result of performing grayscale analysis on A in the image;

[0023] Figure 4 The figure shows the effect of transient overexpression of the TAB2 gene on the replication of RABV CVS11 strain; among them, Figure 4 A in the TCID 50 The figure shows the results of detecting the effect of transient overexpression of the TAB2 gene on the progeny viral titer of the RABV CVS11 strain. Figure 4 Figure B in the graph shows the effect of transient TAB2 gene overexpression on the mRNA transcription level of RABV CVS11 strain detected by RT-qPCR. Figure 4 Figure C in the diagram represents the results of Western blotting analysis on the effect of transient TAB2 gene overexpression on the protein levels of the RABV CVS11 strain. Figure 4 D in the figure represents the pair Figure 4 The result of grayscale analysis of C in the image;

[0024] Figure 5 The figure shows the effect of transient overexpression of the TAB2 gene on the replication of RABV SRV9 strain; among them, Figure 5 A in the TCID 50 The figure shows the effect of transient overexpression of the TAB2 gene on the progeny viral titer of the RABV SRV9 strain. Figure 5 Figure B in the diagram shows the results of RT-qPCR detection of the effect of transient TAB2 gene overexpression on the mRNA transcription level of RABV SRV9 strain. Figure 5 Figure C in the graph represents the results of Western blotting analysis on the effect of transient TAB2 gene overexpression on the protein level of the RABV SRV9 strain. Figure 5 D in the figure represents the pair Figure 5 The result of grayscale analysis of C in the image;

[0025] Figure 6 Figure showing the results of RT-qPCR detection of interference efficiency of three TAB2 gene siRNAs;

[0026] Figure 7 The figure shows the effect of transient TAB2 gene knockdown on the replication of RABV CVS11 strain; among them, Figure 7 A in the TCID 50 The figure shows the results of detecting the effect of transient TAB2 gene knockdown on the progeny viral titer of the RABV CVS11 strain. Figure 7 Figure B in the graph shows the effect of RT-qPCR detection on the mRNA transcription level of the RABV CVS11 strain of the TAB2 gene transient knockdown. Figure 7 Figure C in the graph represents the results of Western blotting analysis on the effect of transient TAB2 gene knockdown on the protein levels of the RABV CVS11 strain. Figure 7 D in the figure represents the pair Figure 7 The result of grayscale analysis of C in the image;

[0027] Figure 8 The figure shows the effect of transient TAB2 gene knockdown on the replication of RABV SRV9 strain; among them, Figure 8 A in the TCID 50 The figure shows the effect of transient TAB2 gene knockdown on the progeny viral titer of the RABV SRV9 strain. Figure 8 Figure B in the graph shows the results of RT-qPCR detection of the effect of transient TAB2 gene knockdown on the mRNA transcription level of RABV SRV9 strain. Figure 8 Figure C in the graph represents the results of Western blotting analysis on the effect of transient TAB2 gene knockdown on the protein levels of the RABV SRV9 strain. Figure 8 D in the figure represents the pair Figure 8 The result of grayscale analysis of C in the image;

[0028] Figure 9 The image shows the results of Western blotting detection of endogenous TAB2 protein in N2a wild-type and knockout cell lines.

[0029] Figure 10 The figure shows the effect of stable TAB2 gene knockout on RABV replication; among them, Figure 10 A in the TCID 50 The figure shows the results of detecting the effect of stable TAB2 gene knockout on the titer of RABV progeny. Figure 10Figure B in the graph shows the results of RT-qPCR detection of the effect of stable TAB2 gene knockout on RABV mRNA transcription levels. Figure 10 Figure C in the graph represents the results of Western blotting analysis on the effect of stable TAB2 gene knockout on RABV protein expression levels. Figure 10 D in the figure represents the pair Figure 10 The result of grayscale analysis of C in the image;

[0030] Figure 11 The figure shows the effect of TAB2 gene reinjection on RABV replication in TAB2-deficient cell lines; among them, Figure 11 A in the TCID 50 The effect of TAB2 protein reintroduction on RABV progeny titers in N2a-ΔTAB2 cells is shown in the figure. Figure 11 Figure B in the graph represents the effect of RT-qPCR on RABV mRNA transcription levels after TAB2 protein reintroduction in N2a-ΔTAB2 cells. Figure 11 In the figure, C represents the results of Western blotting analysis on the effect of TAB2 protein reintroduction on RABV protein levels in N2a-ΔTAB2 cells. Figure 11 D in the figure represents the pair Figure 11 The result of grayscale analysis of C in the image;

[0031] Figure 12 This is a schematic diagram of the recombinant plasmids containing the structural proteins of RABV; among them, Figure 12 A in the diagram represents the pcDNA3.1-Flag-RABV N recombinant plasmid. Figure 12 B in the diagram is a schematic diagram of the pcDNA3.1-Flag-RABV P recombinant plasmid; Figure 12 The C in the diagram represents the pcDNA3.1-Flag-RABV M recombinant plasmid. Figure 12 The D in the diagram represents the pcDNA3.1-Flag-RABV G recombinant plasmid.

[0032] Figure 13 The figure shows the results of the interaction between TAB2 protein and RABV M protein; where, Figure 13 A in the diagram represents the interaction structure between the TAB2 protein and various structural proteins of RABV. Figure 13 In the diagram, B represents the result of co-localization of TAB2 and RABVM proteins within the cell. Figure 13 C in the diagram represents the RABVM protein truncation strategy. Figure 13 In the diagram, D represents the interaction results between the various truncated forms of RABVM and the TAB2 protein. Figure 13 E in the diagram represents the truncation strategy of the TAB2 protein. Figure 13 F in the figure represents the interaction results between the various truncated TAB2 variants and the RABV M protein;

[0033] Figure 14 The diagram shows the results of how the TAB2 protein affects the key structural domains of RABV replication; among them, Figure 14 Figure A shows the effect of reintroducing different TAB2 domains on RABV mRNA transcription levels in the N2a-ΔTAB2 cell line. Figure 14 In the figure, B represents the results of Western blotting analysis of key structural domains that influence the level of RABV protein by TAB2. Figure 14 C in the text is a pair Figure 14 The result of performing grayscale analysis on B in the image;

[0034] Figure 15 The figure shows the effect of TAB2 protein on the MAPK signaling pathway during RABV infection; where, Figure 15 Figure A in the figure shows the results of Western blotting analysis on the effects of transient overexpression or knockdown of the TAB2 gene followed by RABV infection on proteins related to the MAPK signaling pathway. Figure 15 B in the text is a pair Figure 15 The result of performing grayscale analysis on A in the image. Figure 15 Figure C in the figure represents the results of Western blotting analysis on the effects of TAB2 gene reinstatement and RABV infection on MAPK signaling pathway-related proteins in TAB2-deficient cell lines. Figure 15 D in the figure represents the pair Figure 15 The result of grayscale analysis of C in the image;

[0035] Figure 16 The figure shows the effect of the p38 inhibitor Gossypetin on TAB2-promoted RABV replication; among them, Figure 16 Figure A shows the effect of different concentrations of the p38 inhibitor Gossypetin on the activity of N2a cells. Figure 16 Figure B in the figure represents the results of Western blotting analysis on the effects of different concentrations of the p38 inhibitor Gossypetin on MAPK signaling pathway-related proteins and RABV proteins after treatment with RABV. Figure 16 Figure C in the figure represents the results of Western blotting analysis on the effects of transient TAB2 gene overexpression on MAPK signaling pathway-related proteins and RABV proteins after RABV infection and treatment with the p38 inhibitor Gossypetin. Figure 16 D and Figure 16 E in the figure represents the pair Figure 18 The result of grayscale analysis of C in the image;

[0036] Figure 17 The figure shows the effect of the JNK inhibitor BSJ-04-122 on the promotion of RABV replication by TAB2; where, Figure 17 Figure A shows the effect of different concentrations of the JNK inhibitor BSJ-04-122 on the activity of N2a cells. Figure 17 Figure B in the figure represents the results of Western blotting analysis to detect the effects of different concentrations of the JNK inhibitor BSJ-04-122 on MAPK signaling pathway-related proteins and RABV proteins after treatment with RABV. Figure 17 Figure C in the figure represents the results of Western blotting analysis on the effects of transient TAB2 gene overexpression on MAPK signaling pathway-related proteins and RABV proteins after RABV infection and treatment with the JNK inhibitor BSJ-04-122. Figure 17 D and Figure 17 E in the figure represents the pair Figure 17 The result of grayscale analysis of C in the image;

[0037] Figure 18 The figure shows the effect of the TAK1 inhibitor Takinib on the promotion of RABV replication by TAB2; where, Figure 18 Figure A shows the effect of different concentrations of the TAK1 inhibitor Takinib on the activity of N2a cells. Figure 18 Figure B in the figure represents the results of Western blotting analysis to detect the effects of different concentrations of the TAK1 inhibitor Takinib on MAPK signaling pathway-related proteins and RABV proteins after treatment with RABV. Figure 18 Figure C in the figure represents the results of Western blotting analysis on the effects of transient TAB2 gene overexpression on MAPK signaling pathway-related proteins and RABV proteins after RABV infection and treatment with the TAK1 inhibitor Takinib. Figure 18 D in Figure 18 E in Figure 18 F and Figure 18 G in the figure represents the pair Figure 18 The result of performing grayscale analysis on C in the image. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the spirit of the contents disclosed in this invention will be described in detail below. Any person skilled in the art who understands the embodiments of this invention can make changes and modifications based on the techniques taught in this invention without departing from the spirit and scope of this invention.

[0039] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, enzymes, cells, plasmids, etc. used are all commercially available unless otherwise specified.

[0041] Example 1: Effects of RABV infection on the expression of host factor TAB2

[0042] (1) CVS11 rabies virus infection experiment

[0043] The N2a cell line (mouse neuroblastoma-producing cells) was counted and divided into 5 × 10⁻⁶ cells. 5 Cells were seeded per well in 6-well plates and incubated at 37°C for 20 h. Then, CVS11 was used to infect N2a cells with an MOI of 0.1. After 1 h of incubation at 37°C, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. At 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h post-infection, 500 μL of PBS was added to each well for washing, followed by 500 μL of pre-chilled PBS. Cells were collected in 1.5 mL EP tubes using a cell scraper. The cells were centrifuged at 3500 rpm for 10 min at 4°C, the supernatant was discarded, and the cell pellet was stored at -80°C for RNA or protein extraction.

[0044] (2) Effect of RABV infection on the transcriptional level of host factor TAB2 mRNA detected by RT-qPCR

[0045] Add 500 μL of Trizol to each cell sample and incubate at room temperature for about 10 minutes to allow for complete cell lysis. Then add 100 μL of pre-chilled chloroform, mix thoroughly by inverting, incubate on ice for 10 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the clear, transparent uppermost layer to a new 1.5 mL EP tube, add an equal volume of pre-chilled isopropanol, mix thoroughly by inverting, incubate on ice for 15 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. A white precipitate will be visible. Discard the supernatant, add 1 mL of 75% ethanol (diluted with DEPC water), centrifuge at 12,000 rpm for 5 minutes at 4°C, repeat twice, remove any residual ethanol from the bottom of the tube, and air dry on ice. Add an appropriate amount of DEPC water to each EP tube, vortex until the precipitate is completely dissolved, and determine the RNA concentration.

[0046] The RNA was diluted to 200 ng / μL using DEPC water and then reverse transcribed to obtain cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, catalog number RR047A) (see the kit's instructions for reaction conditions and system).

[0047] Using cDNA as a template, PowerUp TM SYBR TM qPCR was performed using Green premixed buffer (applied biosystems, catalog number A25742) (see the kit's instructions for reaction conditions and systems). The primer sequences for TAB2 and β-Actin are shown in Table 1.

[0048] Table 1. Primer sequence information for TAB2 and β-Actin RT-qPCR

[0049]

[0050] RT-qPCR results showed that after RABV CVS11 strain infected N2a cells, as the virus proliferated, the TAB2 mRNA transcription level in the virus-infected group was significantly increased compared with the uninfected group (see [link to RT-qPCR results]). Figure 1 (A) in the middle.

[0051] (3) Western blotting to detect the effect of RABV infection on the expression level of host factor TAB2 protein

[0052] Add 100 μL of cell lysis buffer containing 1% PMSF to each cell sample, vortex until no precipitate forms, and incubate on ice for 10 min; centrifuge at 12000 rpm for 15 min at 4℃, and collect the supernatant as total protein. Protein expression was detected using Western blotting with Anti-TAB2 antibody (abcam, catalog number ab264309). Intracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control gene, and GAPDH polyclonal antibody (Proteintech, catalog number 10494-1-AP) was used (see...). Figure 1 (B in the text). Gray-scale analysis showed that after RABV CVS11 strain infected N2a cells, with viral proliferation, the expression level of TAB2 protein in the virus-infected group was significantly increased compared with the uninfected group (see [reference]). Figure 1 (C in the middle).

[0053] Example 2: Effect of transient TAB2 gene overexpression on RABV CVS11 strain replication

[0054] (1) Construction of TAB2 protein expression vector

[0055] The nucleotide sequence of the mouse TAB2 protein has the NCBI accession number NM_001359534.1, and the amino acid sequence has the NCBI accession number NP_001346463.1. Based on the nucleotide sequence of the mouse TAB2 protein, the full-length upstream primer musTAB2-F (with an added NheⅠ restriction site) and the downstream primer musTAB2-R (with an added HindⅢ restriction site) were designed and synthesized. The primer sequences are shown in Table 2. Using total RNA extracted from N2a cells as a template, the target fragment TAB2 was amplified. The target band was separated by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit (OMEGA, catalog number D2500-01). The pcDNA3.1 vector and the TAB2 target gene fragment were digested with Thermo's rapid restriction endonucleases NheⅠ and HindⅢ at 37℃ for 2 h, respectively. The digested fragments were then purified using the AxyPrep PCR Clean Kit (Axygen, catalog number AP-PCR-50). The pcDNA3.1 double digestion product and the TAB2 target gene fragment were ligated using T4 DNA ligase (New England BioLabs, catalog number M0202L) according to the manufacturer's instructions. The ligation was then performed on DH5α competent cells, and single clones were selected for sequencing. Finally, the fragment was inserted into the pcDNA3.1 vector. A schematic diagram of the pcDNA3.1-musTAB2 recombinant plasmid is shown below. Figure 2 As shown.

[0056] Table 2. Full-length primer sequence information for TAB2

[0057]

[0058] After the recombinant plasmid was correctly sequenced, 1 μg of pcDNA3.1-musTAB2 was taken and processed by PolyJet. TM N2a cells were transfected with in vitro DNA transfection reagent. Cell lysates were collected 48 hours later. Protein expression was detected using Western blotting with anti-TAB2 antibody (Abcam, catalog number ab264309). Intracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control gene, and GAPDH polyclonal antibody (Proteintech, catalog number 10494-1-AP) was used. Western blotting results are shown below. Figure 3 A in the figure. Gray-scale analysis showed that TAB2 protein expression was good (see Figure A). Figure 3 (B in the middle).

[0059] (2) Infection experiment with RABV CVS11 strain after transient overexpression of TAB2 protein

[0060] The N2a cell line was counted and set at 3.5 × 10⁻⁶. 5 pcDNA3.1-musTAB2 was seeded per well in a 6-well plate and incubated at 37°C for 20 hours. 1 μg of pcDNA3.1-musTAB2 was then transferred via PolyJet. TM N2a cells were transfected with an in vitro DNA transfection reagent (see the liposome's instructions for transfection conditions and system). 24 h after transfection, N2a cells were infected with CVS11 at an MOI of 0.1. After incubation at 37°C for 1 h, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. Cell supernatant and cell pellet were collected at 24 h, 36 h, and 48 h post-infection and stored at -80°C.

[0061] (3)TCID 50 Detecting the effect of transient overexpression of the TAB2 gene on the titer of RABV progeny viruses

[0062] The viral supernatant to be tested was serially diluted 9 times at a 10-fold gradient to a final concentration of 10. -9 Dilution was determined, and N2a cells that had grown to a confluent monolayer were passaged and seeded at 100 μL / well in 96-well plates. 10 -9 ~10 -1 Virus dilutions of different concentrations were added sequentially to 96-well plates at 50 μL / well, with four replicates for each dilution. Cells and virus solution were thoroughly mixed and incubated at 37°C for 48 h. After 48 h, the supernatant was discarded, and 150 μL / well of pre-chilled 80% acetone was added, with fixation at room temperature for 30 min. The fixative was discarded, and 150 μL / well of PBST was added. The plates were washed, and this step was repeated three times, with the last wash performed by gently tapping the plate on paper to remove the liquid from the wells. 50 μL / well of diluted fluorescent antibody was added, and the plates were incubated at 37°C in the dark for 1 h. The supernatant was discarded, and 150 μL / well of PBST was added. The plates were washed, and this step was repeated three times, with the last wash performed by gently tapping the plate on paper to remove the liquid from the wells. The 96-well plates were observed under a fluorescence microscope, and the number of positive wells for each dilution was recorded. TCID was calculated. 50 .

[0063] The results showed that compared with the group transfected with pcDNA3.1 plasmid, the RABV progeny virus titer was significantly increased in the group transfected with pcDNA3.1-musTAB2 plasmid, indicating that TAB2 protein overexpression can significantly promote the proliferation of RABV CVS11 strain in N2a cells (see [link to study]). Figure 4 (A) in the middle.

[0064] (4) RT-qPCR detection of the effect of transient TAB2 gene overexpression on RABV mRNA transcription level

[0065] The transcriptional level of RABV N protein mRNA was detected by RT-qPCR. The specific steps are shown in Example 1. The primer sequences for RABV N RT-qPCR are shown in Table 3.

[0066] Table 3. Primer sequence information for RABV N RT-qPCR

[0067]

[0068] RT-qPCR results showed that compared with the group transfected with pcDNA3.1 plasmid, the group transfected with pcDNA3.1-musTAB2 plasmid had significantly increased RABV N protein mRNA transcription levels, indicating that TAB2 protein overexpression can significantly promote the replication of RABV CVS11 strain in N2a cells (see [link to RT-qPCR results]). Figure 4 (B in the middle).

[0069] (5) Western blotting was used to detect the effect of transient TAB2 gene overexpression on RABV protein levels.

[0070] RABV N protein expression was detected by Western blotting using an anti-RABV N antibody (prepared and preserved in our laboratory). Specific steps are detailed in Example 1, and results are shown below. Figure 4 C. Gray-scale analysis showed that, compared with the group transfected with pcDNA3.1 plasmid, the expression level of RABV N protein in the group transfected with pcDNA3.1-musTAB2 plasmid was significantly increased, indicating that TAB2 protein overexpression can significantly promote the replication of RABV CVS11 strain in N2a cells (see C). Figure 4 (D in the middle).

[0071] Example 3: Effect of transient TAB2 gene overexpression on RABV SRV9 strain replication

[0072] (1) Infection experiment with RABV SRV9 strain after transient overexpression of TAB2 protein

[0073] The N2a cell line was counted and set at 3.5 × 10⁻⁶. 5 pcDNA3.1-musTAB2 was seeded per well in a 6-well plate and incubated at 37°C for 20 hours. 1 μg of pcDNA3.1-musTAB2 was then transferred via PolyJet. TM N2a cells were transfected with an in vitro DNA transfection reagent (see the liposome's instructions for transfection conditions and system). 24 h after transfection, N2a cells were infected with SRV9 at MOI = 0.1. After incubation at 37°C for 1 h, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. Cell supernatant and cell pellet were collected at 24 h, 36 h, and 48 h post-infection, and stored at -80°C.

[0074] (2)TCID 50 Detecting the effect of transient overexpression of the TAB2 gene on the titer of RABV progeny viruses

[0075] Using TCID 50 The RABV viral titer was detected, and the specific steps are described in Example 2. The results showed that compared with the group transfected with pcDNA3.1 plasmid, the RABV progeny viral titer was significantly increased in the group transfected with pcDNA3.1-musTAB2 plasmid, indicating that TAB2 protein overexpression can significantly promote the proliferation of RABV SRV9 strain in N2a cells (see Example 2). Figure 5 (A) in the middle.

[0076] (3) RT-qPCR detection of the effect of transient TAB2 gene overexpression on RABV mRNA transcription level

[0077] The transcriptional level of RABV N protein mRNA was detected using RT-qPCR, with specific steps described in Example 1. Results showed that compared to the group transfected with pcDNA3.1 plasmid, the transcriptional level of RABV N protein mRNA was significantly increased in the group transfected with pcDNA3.1-musTAB2 plasmid, indicating that TAB2 protein overexpression can significantly promote the replication of RABV SRV9 strain in N2a cells (see Example 1). Figure 5 (B in the middle).

[0078] (4) Western blotting to detect the effect of transient TAB2 gene overexpression on RABV protein levels

[0079] The expression level of RABV N protein was detected by Western blotting. Specific steps are described in Example 1, and the results are shown below. Figure 5 C. Gray-scale analysis showed that, compared with the group transfected with pcDNA3.1 plasmid, the expression level of RABV N protein in the group transfected with pcDNA3.1-musTAB2 plasmid was significantly increased, indicating that TAB2 protein overexpression can significantly promote the replication of RABV SRV9 strain in N2a cells (see [link to study].) Figure 5 (D in the middle).

[0080] Example 4: Effect of transient TAB2 gene knockdown on RABV CVS11 strain replication

[0081] (1) Interference efficiency of siRNA

[0082] Three synthesized TAB2 gene siRNAs (sequence information shown in Table 4) were transfected into N2a cells and co-transfected (with a siNC control also included). TAB2 mRNA transcription levels were detected by RT-qPCR. The results showed that all three siRNAs could inhibit TAB2 gene expression (see Table 4). Figure 6 To ensure the interference effect, subsequent experiments selected to mix the three siRNAs in a 1:1:1 ratio before transfecting the cells.

[0083] Table 4. TAB2 siRNA sequence information

[0084]

[0085] (2) Infection experiment with RABV CVS11 strain after transient knockdown of TAB2 protein

[0086] The N2a cell line was counted and set at 3.5 × 10⁻⁶. 5 Seeds were seeded per well in 6-well plates. After incubation at 37°C for 20 hours, the samples were analyzed using Lipofectamine. TM The TAB2 siRNA obtained from the above screening was transfected into N2a cells using RNAiMAX transfection reagent (transfection conditions and system are described in the liposome's instructions). 24 h after transfection, N2a cells were infected with CVS11 at MOI = 0.1. After incubation at 37°C for 1 h, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. Cell supernatant and cell pellet were collected at 24 h, 36 h, and 48 h post-infection, and stored at -80°C.

[0087] (3)TCID 50 Detecting the effect of transient TAB2 gene knockdown on RABV progeny viral titers

[0088] Using TCID 50 The RABV viral titer was detected, and the specific steps are described in Example 2. The results showed that compared with the siRNA-NC transfected group, the RABV progeny viral titer in the siRNA-TAB2 transfected group was significantly lower, indicating that TAB2 knockdown expression can significantly inhibit the proliferation of the RABV VVS11 strain in N2a cells (see Example 2). Figure 7 (A) in the middle.

[0089] (4) RT-qPCR detection of the effect of transient TAB2 gene knockdown on RABV mRNA transcription level

[0090] The transcriptional level of RABV N protein mRNA was detected using RT-qPCR, with specific steps described in Example 1. Results showed that the transcriptional level of RABV N protein mRNA was significantly lower in the siRNA-TAB2 transfected group compared to the siRNA-NC transfected group, indicating that TAB2 protein knockdown significantly inhibited the replication of RABV CVS11 strain in N2a cells (see Example 1). Figure 7 (B in the middle).

[0091] (5) Western blotting to detect the effect of transient TAB2 gene knockdown on RABV protein levels

[0092] The expression level of RABV N protein was detected by Western blotting. Specific steps are described in Example 1, and the results are shown below. Figure 7 C. Gray-scale analysis showed that, compared with the siRNA-NC group, the siRNA-TAB2 group had a significantly lower expression level of RABV N protein, indicating that TAB2 protein knockdown can significantly inhibit the replication of RABV CVS11 strain in N2a cells (see C). Figure 7 (D in the middle).

[0093] Example 5: Effect of transient TAB2 gene knockdown on RABV SRV9 strain replication

[0094] (1) Infection experiment with RABV SRV9 strain after transient knockdown of TAB2 protein

[0095] The N2a cell line was counted and set at 3.5 × 10⁻⁶. 5 Seeds were seeded per well in 6-well plates. After incubation at 37°C for 20 hours, the samples were analyzed using Lipofectamine. TM The TAB2 siRNA obtained from the above screening was transfected into N2a cells using RNAiMAX transfection reagent (transfection conditions and system are described in the liposome's instructions). 24 h after transfection, N2a cells were infected with SRV9 at MOI = 0.1. After incubation at 37°C for 1 h, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. Cell supernatant and cell pellet were collected at 24 h, 36 h, and 48 h post-infection, and stored at -80°C.

[0096] (2)TCID 50 Detecting the effect of transient TAB2 gene knockdown on RABV progeny viral titers

[0097] Using TCID 50 The RABV viral titer was detected, and the specific steps are described in Example 2. The results showed that compared with the siRNA-NC transfected group, the RABV progeny viral titer in the siRNA-TAB2 transfected group was significantly lower, indicating that TAB2 knockdown expression can significantly inhibit the proliferation of RABV SRV9 strain in N2a cells (see Example 2). Figure 8 (A) in the middle.

[0098] (3) RT-qPCR detection of the effect of transient TAB2 gene knockdown on RABV mRNA transcription level

[0099] The transcriptional level of RABV N protein mRNA was detected using RT-qPCR, with specific steps described in Example 1. Results showed that compared to the siRNA-NC transfected group, the siRNA-TAB2 transfected group exhibited significantly reduced RABV N protein mRNA transcription, indicating that TAB2 protein knockdown significantly inhibits the replication of RABV SRV9 strain in N2a cells (see Example 1). Figure 8 (B in the middle).

[0100] (4) Western blotting to detect the effect of transient TAB2 gene knockdown on RABV protein levels

[0101] The expression level of RABV N protein was detected by Western blotting. Specific steps are described in Example 1, and the results are shown below. Figure 8 C. Gray-scale analysis showed that, compared with the siRNA-NC group, the siRNA-TAB2 group had significantly lower RABV N protein expression levels, indicating that TAB2 protein knockdown can significantly inhibit the replication of RABV SRV9 strain in N2a cells (see [link to data]). Figure 8 (D in the middle).

[0102] Example 6: The effect of stable TAB2 gene knockout on RABV replication

[0103] (1) Construction of TAB2 gene deletion cell line

[0104] We used CRISPR-Cas9 technology to construct cell lines. Based on the mouse TAB2 reference nucleotide sequence (Mus musculus, NM_001359534.1) published by NCBI, we designed sgRNAs targeting this protein using online software http: / / crispr.mit.edu / (sequence information is shown in Table 5).

[0105] Table 5 musTAB2-sgRNA sequence information

[0106]

[0107] Using the pMD18T-U6-huANPsgRNA-1 recombinant plasmid (see Chinese patent application CN201810177710.X, entitled "Application of ANP32 Protein in Maintaining Influenza Virus Polymerase Activity in the Host") retained in the laboratory as a template, primers for sgRNA targeting musTAB2 were designed. The plasmid containing musTAB2 sgRNA was constructed by amplification using KOD-FXNeo high-efficiency DNA polymerase (catalog number: KFX-201, purchased from Toyobo) using the overlap PCR method (reaction conditions and system are described in the polymerase instructions). Primer sequences are shown in Table 6. The PCR system and procedure were performed in accordance with the KOD-FX Neo instruction manual. The obtained PCR product was digested with DpnI in a 37°C water bath for 30 minutes. Then, 5 μl of the digested product was transformed into 20 μl of DH5α competent cells. The next day, single clones were picked and sequenced. The plasmids with correct sequencing, namely pMD18T-U6-musTAB2sgRNA-1 or pMD18T-U6-musTAB2sgRNA-2 (both containing musTAB2sgRNA), were used for subsequent transfection experiments.

[0108] Table 6. Primer sequence information for musTAB2-sgRNA

[0109]

[0110] One μg each of the eukaryotic plasmid pMJ920 (Addgene plasmid #42234) expressing Cas9-GFP protein and the recombinant plasmids pMD18T-U6-musTAB2sgRNA-1 and pMD18T-U6-musTAB2sgRNA-2 were analyzed by PolyJet. TM N2a cells were transfected with an in vitro DNA transfection reagent. Forty-eight hours later, GFP-positive cells were screened using an ultraflow cytometry system and seeded as single cells per well in 96-well plates. After approximately 10 days, single-cell clones were picked and cultured. Identification was performed using Western blotting; the specific steps are described in Example 1. Results are shown in [Figure 1]. Figure 9 .Depend on Figure 9 The results show that the present invention successfully constructed a musTAB2 single knockout cell line (N2a-ΔTAB2) and used it for subsequent experiments.

[0111] (2) Infection experiment with RABV CVS11 strain after stable knockout of TAB2 protein

[0112] Wild-type N2a cell lines and N2a-ΔTAB2 cell lines were counted and divided into groups of 3.5 × 10⁻⁶. 5Cells were seeded per well in 6-well plates. After incubation at 37°C for 20 h, CVS11 was used to infect different cell lines with an MOI of 0.1. After 1 h of incubation at 37°C, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. Cell supernatant and cell pellet were collected at 24 h, 36 h, and 48 h post-infection and stored at -80°C.

[0113] (3)TCID 50 Detecting the effect of stable TAB2 gene knockout on RABV progeny viral titers

[0114] Using TCID 50 The RABV viral titer was determined, and the specific steps are described in Example 2. The results showed that the RABV progeny viral titer was significantly lower in N2a-ΔTAB2 cells compared to wild-type N2a cells, indicating that TAB2 protein knockout significantly inhibited RABV proliferation in N2a cells (see Example 2). Figure 10 (A) in the middle.

[0115] (4) RT-qPCR detection of the effect of stable TAB2 gene knockout on RABV mRNA transcription level

[0116] The transcriptional level of RABV N protein mRNA was detected using RT-qPCR, with specific procedures described in Example 1. Results showed that the transcriptional level of RABV N protein mRNA was significantly reduced in N2a-ΔTAB2 cells compared to wild-type N2a cells, indicating that TAB2 protein knockout significantly inhibits RABV replication in N2a cells (see Example 1). Figure 10 (B in the middle).

[0117] (5) Western blotting to detect the effect of stable TAB2 gene knockout on RABV protein levels

[0118] The expression level of RABV N protein was detected using Western blotting. Specific steps are described in Example 1, and the results are shown below. Figure 10 C. Gray-scale analysis showed that, compared with wild-type N2a cells, the expression level of RABV N protein in N2a-ΔTAB2 cells was significantly reduced, indicating that TAB2 protein knockout can significantly inhibit RABV replication in N2a cells (see [reference needed]). Figure 10 (D in the middle).

[0119] Example 7: Effect of TAB2 gene reinjection on RABV replication in TAB2-deficient cell lines

[0120] (1) CVS11 rabies virus infection experiment after TAB2 protein replenishment

[0121] Wild-type N2a cell lines and N2a-ΔTAB2 cell lines were counted and divided into groups of 3.5 × 10⁻⁶. 5 Each plasmid was seeded into a 6-well plate. After incubation at 37°C for 20 hours, 1 μg of recombinant plasmid pcDNA3.1-musTAB2 (constructed in Example 2) was extracted and processed using PolyJet. TM N2a-ΔTAB2 cells were transfected with an in vitro DNA transfection reagent (to obtain N2a-ΔTAB2 cells replenished with TAB2 protein, denoted as N2a-ΔTAB2+TAB2). 1 μg of empty vector plasmid pcDNA3.1 was then transfected using PolyJet. TM N2a cells were transfected with an in vitro DNA transfection reagent (to obtain wild-type N2a cells containing empty vectors, denoted as N2a). 24 h after transfection, different cell types (N2a, N2a-ΔTAB2, and N2a-ΔTAB2+TAB2) were infected with CVS11 at an MOI of 0.1. After incubation at 37°C for 1 h, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. Cell supernatant and cell pellet were collected 48 h post-infection and stored at -80°C.

[0122] (2)TCID 50 Examining the effect of TAB2 protein reintroduction in N2a-ΔTAB2 cells on RABV progeny viral titers.

[0123] Using TCID 50 The RABV viral titer was detected, and the specific steps are described in Example 2. The results showed that the RABV progeny viral titer significantly increased after TAB2 was reintroduced into N2a-ΔTAB2 cells, indicating that TAB2 protein reintroduction could relieve the inhibition of RABV proliferation in N2a-ΔTAB2 cells (see Example 2). Figure 11 (A) in the middle.

[0124] (2) RT-qPCR detection of the effect of TAB2 protein reintroduction on RABV mRNA transcription level in N2a-ΔTAB2 cells.

[0125] The transcriptional level of RABV N protein mRNA was detected using RT-qPCR, with specific procedures described in Example 1. Results showed that the transcriptional level of RABV N protein mRNA significantly increased after TAB2 was reintroduced into N2a-ΔTAB2 cells, indicating that TAB2 protein reintroduction could restore the promoting effect on RABV replication (see Example 1). Figure 11 (B in the middle).

[0126] (4) Western blotting was used to detect the effect of TAB2 protein refilling on RABV protein levels in N2a-ΔTAB2 cells.

[0127] The expression level of RABV N protein was detected by Western blotting. Specific steps are described in Example 1, and the results are shown below. Figure 11 C. Gray-scale analysis showed that the expression level of RABV N protein significantly increased after TAB2 was reintroduced into N2a-ΔTAB2 cells, indicating that TAB2 protein reintroduction can relieve the inhibition of RABV replication in N2a-ΔTAB2 cells (see C). Figure 11 (D in the middle).

[0128] Example 8: Interaction between TAB2 protein and RABV M protein

[0129] (1) Interaction between TAB2 protein and various structural proteins of RABV

[0130] The nucleotide sequence of the mouse TAB2 protein has the NCBI accession number NM_001359534.1, and the amino acid sequence has the NCBI accession number NP_001346463.1. Based on the nucleotide sequence of the mouse TAB2 protein, the full-length upstream primer TAB2-F' (with an EcoRI restriction site added) and the downstream primer TAB2-R' (with a BamHI restriction site added) were designed and synthesized. The primer sequences are shown in Table 7. Using N2a cells as a template, the target fragment TAB2 was amplified. The target band was separated by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit (OMEGA, catalog number D2500-01). The pcDNA3.0-Flag, pcDNA3.0-HA vector, and TAB2 target gene fragment were digested with Thermo's rapid restriction endonucleases EcoRI and BamHI at 37°C for 2 h, respectively. The digested fragments were then purified using the AxyPrep PCR Cleaning Kit (Axygen, catalog number AP-PCR-50). The pcDNA3.0-Flag or pcDNA3.0-HA double digestion products were ligated to the TAB2 target gene fragment using T4 DNA ligase (New England BioLabs, catalog number M0202L) according to the manufacturer's instructions. The ligation was then performed on DH5α competent cells, and single clones were selected for sequencing. Finally, the fragments were inserted into the pcDNA3.0-Flag or pcDNA3.0-HA vector.

[0131] Table 7. Full-length primer sequence information for TAB2

[0132]

[0133] After confirming the recombinant plasmid sequencing was correct, 1 μg of pcDNA3.0-HA-musTAB2 was used to recombinant with the following eukaryotic expression plasmids for RABV structural proteins: 1 μg pcDNA3.0-Flag-RABV N, 1 μg pcDNA3.0-Flag-RABV P, 1 μg pcDNA3.0-Flag-RABV VM, and 1 μg pcDNA3.0-Flag-RABV G (see plasmid map). Figure 12 via PolyJet TM In vitro DNA transfection reagent was co-transfected into 293T cells (human embryonic kidney cells), and a negative control was set up. After 48 hours, cell lysates were collected, a portion of the supernatant was reserved as an input control, and the remaining supernatant was incubated overnight at 4°C with Anti-Flag Magnetic Beads (MedChemExpress, catalog number HY-K0207) (washed with PBS). The beads were then adsorbed onto a magnetic rack, the supernatant was discarded, and the beads were resuspended in 1 mL of PBS. The supernatant was discarded, and the beads were washed 5 times. The beads were then resuspended in 1×SDS PAGE loading buffer. Protein expression was detected using Western blotting with Anti-Flag antibody (Cell Signaling Technology, catalog number 14793S) and Anti-HA antibody (Thermo Scientific, catalog number MA5-27915). Specific steps are described in Example 1, and the results are shown below. Figure 13 The result from A indicates that TAB2 interacts only with RABVM within the cell.

[0134] (2) TAB2 protein and RABVM protein co-localize in cells

[0135] N2a cells were passaged normally into 35 mm confocal microplates at a cell density of 3 × 10⁻⁶ cells / mL. 4After culturing at 37°C for 20 h at a concentration of [number] cells / mL, N2a cells were transfected individually or co-transfected with the eukaryotic expression plasmids pcDNA3.0-Flag-RABV M (expressing RABV M protein) and pcDNA3.0-HA-musTAB2 (expressing TAB2 protein). 24 h post-transfection, the supernatant was discarded, and cells were fixed in each well with 300 μL of 4% paraformaldehyde at room temperature for 30 min. The supernatant was discarded, and cells were washed three times with PBS, 5 min each time. Cells were permeated in each well with 300 μL of 0.2% Triton-X-100-PBS at room temperature for 5 min. The supernatant was discarded, and cells were washed three times with PBS, 5 min each time. Cells were blocked in each well with 500 μL of 1% BSA in PBS at room temperature for 30 min or overnight at 4°C. Subsequently, the primary antibody was diluted with blocking buffer and incubated in 200 μL per well at 37°C for 1 h. Cells were washed three times with PBST, 5 min each time. Dilute the secondary antibody with blocking buffer, 200 μL per well, and incubate at 37°C in the dark for 1 h. Wash three times with PBST, 5 min each time. Place one drop of DAPI-containing antifluorescence quencher in the center of the dish and observe after 3-5 min. Then, add one drop of 90% glycerol to the center of the dish, cover with a coverslip, and store at 4°C in the dark. Confocal analysis revealed that TAB2 protein and RABV M protein co-localize in cells (see...). Figure 13 (B in the middle).

[0136] (3) The structural domains of RABVM and TAB2 interaction

[0137] The nucleotide sequence of RABV is accessed by NCBI under accession number MN599474.1, and the amino acid sequence of the RABV M protein is accessed by NCBI under accession number QXN53096.1. The RABV M truncation strategy is as follows: Figure 13 As shown in C, the upstream primer RABVM, a truncated form of RABVM, was designed and synthesized based on the nucleotide sequence of RABVM. 1-202 -F, RABVM 1-76 -F, RABVM 77-202 -F (with added KpnⅠ restriction site) and downstream primer RABVM 1-202 -R、RABV M 1-76 -R、RABV M 77-202-R (addition of XhoⅠ restriction site), primer sequences are shown in Table 8. Using pcDNA3.0-Flag-RABV M plasmid as a template, we amplified the target fragment RABVM. The target band was separated by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit (OMEGA, catalog number D2500-01). The pCAGGS-GST vector and the RABV M target gene fragment were digested with Thermo rapid restriction endonucleases KpnⅠ and XhoⅠ at 37℃ for 2 h, and then purified using an AxyPrep PCR cleaning kit (Axygen, catalog number AP-PCR-50). The pCAGGS-GST double digestion product was ligated to the TAB2 target gene fragment using T4 DNA ligase (New England BioLabs, catalog number M0202L) according to the manufacturer's instructions. The ligation was then performed on DH5α competent cells, and single clones were selected for sequencing. Finally, the fragment was inserted into the pCAGGS-GST vector.

[0138] Table 8. Primer sequence information for RABV M truncated form

[0139]

[0140]

[0141] After the recombinant plasmid was correctly sequenced, 1 μg of pcDNA3.0-Flag-musTAB2 was taken and reacted with 1 μg of pCAGGS-GST-RABV M, a truncated eukaryotic expression plasmid of TAB2. 1-202 1μg pCAGGS-GST-RABV M 1-76 1μg pCAGGS-GST-RABVM 77-202 via PolyJet TM In vitro DNA transfection reagent was used to transfect 293T cells, and a negative control was set up. After 48 hours, cell lysates were collected, and a portion of the supernatant was reserved as an input control. The remaining supernatant was incubated overnight at 4°C with Anti-Flag Magnetic Beads (MedChemExpress, catalog number HY-K0207) (washed with PBS). The beads were then adsorbed onto a magnetic rack, the supernatant was discarded, and the beads were resuspended in 1 mL PBS. The supernatant was discarded, and the beads were washed 5 times. The beads were then resuspended in 1×SDS PAGE loading buffer. Protein expression was detected using Western blotting with Anti-Flag antibody (Cell Signaling Technology, catalog number 14793S) and Anti-HA antibody (Thermo Scientific, catalog number MA5-27915). Specific procedures are detailed in Example 1, and results are shown in Appendix I. Figure 13From the result, we can see that the domain through which TAB2 interacts with RABV M is RABV M. 77-202 .

[0142] (4) The structural domains of interaction between TAB2 and RABV M

[0143] The nucleotide sequence of the mouse TAB2 protein has the NCBI accession number NM_001359534.1, and the amino acid sequence has the NCBI accession number NP_001346463.1. The TAB2 truncation strategy is as follows: Figure 13 As shown in E, the truncated upstream primer TAB2 was designed and synthesized based on the nucleotide sequence of mouse TAB2. 1-50 -F、TAB2 51-574 -F、TAB2 575-693 -F (addition of KpnⅠ restriction site) and downstream primer TAB2 1-50 -R、TAB2 51-574 -R、TAB2 575-693 -R (with added EcoRI restriction site), primer sequences are shown in Table 9. Using N2a cells as a template, we amplified the target fragment TAB2. The target band was separated by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit (OMEGA, catalog number D2500-01). The pCAGGS-GST vector and the TAB2 target gene fragment were digested with Thermo rapid restriction endonucleases KpnⅠ and EcoRI at 37℃ for 2 h, respectively. The digested fragments were then purified using an AxyPrep PCR cleaning kit (Axygen, catalog number AP-PCR-50). The pCAGGS-GST double digestion product was ligated to the TAB2 target gene fragment using T4 DNA ligase (New England BioLabs, catalog number M0202L) according to the manufacturer's instructions. The ligation was then performed on DH5α competent cells, and single clones were selected for sequencing. Finally, the fragment was inserted into the pCAGGS-GST vector.

[0144] Table 9. TAB2 truncated primer sequence information

[0145]

[0146] After confirming the correct sequencing of the recombinant plasmid, 1 μg of pcDNA3.0-Flag-RABV M was used to express 1 μg of pcDNA3.0-HA-musTAB2 and 1 μg of pCAGGS-GST-musTAB2 eukaryotic expression plasmids, respectively. 1-50 1μg pCAGGS-GST-musTAB2 51-574 1μg pCAGGS-GST-musTAB2575-693 via PolyJet TM In vitro DNA transfection reagents were co-transfected into 293T cells, with a negative control included. After 48 hours, cell lysates were collected, with a portion of the supernatant reserved as an input control. The remaining supernatant was incubated overnight at 4°C with Anti-Flag Magnetic Beads (MedChemExpress, catalog number HY-K0207) (washed with PBS). The beads were then adsorbed onto a magnetic rack, the supernatant discarded, and the beads resuspended in 1 mL PBS. The supernatant was discarded, and the beads were washed five times. The beads were then resuspended in 1×SDSPAGE loading buffer. Protein expression was detected using Western blotting with Anti-Flag antibody (Cell Signaling Technology, catalog number 14793S), Anti-HA antibody (Thermo Scientific, catalog number MA5-27915), and Anti-GST antibody (Proteintech, catalog number 10000-0-AP). Specific procedures are detailed in Example 1, and results are shown below. Figure 13 From the result, we can see that the domain through which TAB2 interacts with RABVM is musTAB2. 51-574 and musTAB2 575-693 .

[0147] Example 9: TAB2 protein affects key domains of RABV replication

[0148] (1) Infection experiment of RABV CVS11 strain after transient overexpression of different truncated forms of TAB2 protein

[0149] The N2a cell line was counted and set at 3.5 × 10⁻⁶. 5 Seeds were seeded per well in 6-well plates and incubated at 37°C for 20 hours. Then, 1 μg of pcDNA3.0-Flag-musTAB2 and 1 μg of pCAGGS-GST-musTAB2 were taken from each well. 1-50 1μg pCAGGS-GST-musTAB2 51-574 1μg pCAGGS-GST-musTAB2 575-693 (All were constructed in Example 8) via PolyJet TM N2a-ΔTAB2 cells (constructed in Example 6) were transfected with an in vitro DNA transfection reagent (see the liposome's instruction manual for transfection conditions and system). 24 h after transfection, N2a-ΔTAB2 cells were infected with CVS11 at an MOI of 0.1. After incubation at 37°C for 1 h, the viral infection solution was discarded, and fresh 5% fetal bovine serum DMEM medium was added. 48 h post-infection, the cell supernatant and cell pellet were collected and stored at -80°C.

[0150] (2) RT-qPCR detection of the effect of different truncated TAB2 variants on RABV mRNA transcription levels

[0151] The transcriptional level of RABV N protein mRNA was detected using RT-qPCR, with specific steps described in Example 1. Results showed that compared to the group transfected with pcDNA3.1 plasmid, the groups transfected with pcDNA3.0-Flag-musTAB2 plasmid and pCAGGS-GST-musTAB2 plasmid showed significantly higher levels of RABV N protein mRNA transcription. 575-693 The significantly elevated RABV N protein mRNA transcription level in the group indicated that TAB2 575-693 It is a key domain of TAB2 that promotes the replication of RABV in N2a cells (see...). Figure 14 (A) in the middle.

[0152] (3) Western blotting to detect the effect of different truncated forms of TAB2 on RABV protein levels

[0153] The expression level of RABV N protein was detected by Western blotting. Specific steps are described in Example 1, and the results are shown below. Figure 14 B in the sample. Gray-scale analysis showed that compared with the group transfected with pcDNA3.1 plasmid, the groups transfected with pcDNA3.0-Flag-musTAB2 plasmid and pCAGGS-GST-musTAB2 plasmid were significantly more effective. 575-693 The expression level of RABV N protein in the group was significantly increased, indicating that TAB2 575-693 It is a key domain of TAB2 that promotes the replication of RABV in N2a cells (see...). Figure 14 (C in the middle).

[0154] Example 10: Effects of TAB2 protein on the MAPK signaling pathway during RABV infection

[0155] (1) Infection experiment with RABV CVS11 strain after transient overexpression or knockdown of TAB2 protein

[0156] For specific steps, see Examples 2 and 4. The cell pellet was collected 12 hours after infection and stored at -80°C.

[0157] (2) Western blotting to detect the effects of transient overexpression or knockdown of TAB2 gene followed by RABV infection on proteins related to the MAPK signaling pathway.

[0158] Western blotting was used to detect the expression of proteins corresponding to the MAPK signaling pathway using the following antibodies: p38 MAPK Antibody (Cell Signaling Technology; catalog number 9212), Phospho-p38 MAPK Antibody (Affinity; catalog number AF4001), p44 / 42MAPK (Erk1 / 2) Antibody (Cell Signaling Technology; catalog number 9102), Phospho-p44 / 42MAPK (Erk1 / 2) Antibody (Cell Signaling Technology; catalog number 8544), Anti-JNK1+JNK2+JNK3 (abcam; catalog number ab179461), and Anti-phospho-JNK1+JNK2+JNK3 (abcam; catalog number ab124956). Specific steps are detailed in Example 1, and results are shown below. Figure 15 A. Gray-scale analysis showed that TAB2 gene overexpression significantly increased p38 protein phosphorylation, while TAB2 gene knockdown significantly decreased p38 protein phosphorylation. This indicates that during RABV infection, TAB2 protein promotes MAPK signaling pathway phosphorylation, with the most significant effect on the p38 / MAPK signaling pathway (see A). Figure 15 (B in the middle).

[0159] (3) CVS11 rabies virus infection experiment after TAB2 protein replenishment

[0160] For specific steps, see Example 7. The cell pellet was collected 48 hours after infection and stored at -80°C.

[0161] (3) Western blotting was used to detect the effect of TAB2 protein refilling on RABV protein levels in N2a-ΔTAB2 cells.

[0162] Western blotting was used to detect the expression of RABV protein and MAPK signaling pathway proteins. Specific steps are described in Example 1, and the results are shown below. Figure 15 C. Gray-scale analysis showed that after TAB2 reintroduction in N2a-ΔTAB2 cells, the expression levels of RABV N protein, p38 protein phosphorylation, and JNK protein phosphorylation were significantly increased. This indicates that TAB2 protein reintroduction can relieve the inhibition of RABV replication in N2a-ΔTAB2 cells and restore MAPK signaling pathway phosphorylation, with significant effects on the p38 / MAPK and JNK / MAPK signaling pathways (see [link to data]). Figure 15 (D in the middle).

[0163] Example 11: Effect of p38 inhibitor Gossypetin on TAB2-promoted RABV replication

[0164] (1) Effects of different concentrations of p38 inhibitor Gossypetin on N2a cell viability

[0165] The N2a cell line was counted and set at 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and incubated at 37°C for 20 h. The medium was then replaced with 5% fetal bovine serum DMEM containing different concentrations of the p38 inhibitor Gossypetin (100 μL / well). After 24 h of medium replacement, 10 μL of CCK-8 solution was added to each well. After further incubation at 37°C for 1 h, the absorbance of each well was measured at 450 nm using a microplate reader to calculate cell viability. Results showed that the p38 inhibitor Gossypetin at working concentrations of 30 μM and below had no significant cytotoxicity (see...). Figure 16 (A) in the middle.

[0166] (2) Experiments involving RABV CVS11 strain infection after transient overexpression of TAB2 protein and treatment with the p38 inhibitor Gossypetin.

[0167] For specific steps, see Example 2. After 1 hour of infection, the medium was replaced with DMEM containing 5% fetal bovine serum with different concentrations of the p38 inhibitor Gossypetin. The cell pellet was collected 24 hours after infection and stored at -80°C.

[0168] (3) Western blotting was used to detect the effects of different concentrations of the p38 inhibitor Gossypetin on MAPK signaling pathway-related proteins and RABV proteins after treatment with RABV and infection.

[0169] Western blotting was performed using the antibodies Anti-RABV N (prepared and preserved in our laboratory), p38 MAPK Antibody (Cell Signaling Technology, catalog number 9212), and Phospho-p38 MAPK Antibody (Affinity; catalog number AF4001). The antibodies were used to detect the expression of the corresponding MAPK signaling pathway proteins and RABV proteins. Specific procedures are detailed in Example 1, and the results are shown below. Figure 16 In section B, the results showed that treatment with the p38 inhibitor Gossypetin significantly inhibited p38 protein phosphorylation, with 20 μM Gossypetin exhibiting good inhibitory effects (see section B). Figure 16 (B in the middle).

[0170] (4) Western blotting analysis of the effects of transient TAB2 gene overexpression on MAPK signaling pathway-related proteins and RABV proteins after RABV infection with p38 inhibitor Gossypetin.

[0171] Western blotting was used to detect the expression of RABV protein and MAPK signaling pathway proteins. Specific steps are detailed in Example 1, and the results are shown in the figure below. Figure 16 C. Gray-scale analysis showed that TAB2 protein overexpression promoted p38 protein phosphorylation and RABV replication. Treatment with the p38 inhibitor Gossypetin neutralized both the promoting effects of TAB2 protein overexpression on p38 phosphorylation and RABV replication. This indicates that TAB2 protein promotes RABV replication through the p38 / MAPK signaling pathway, and that the p38 inhibitor Gossypetin may be a potential drug for treating RABV infection (see [link to study]). Figure 16 D and Figure 16 (E in the text)

[0172] Example 12: Effect of JNK inhibitor BSJ-04-122 on TAB2-promoted RABV replication

[0173] (1) Effects of different concentrations of JNK inhibitor BSJ-04-122 on N2a cell viability

[0174] The N2a cell line was counted and set at 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and incubated at 37°C for 20 h. The medium was then replaced with 5% fetal bovine serum DMEM containing different concentrations of the JNK inhibitor BSJ-04-122 (100 μL / well). After 24 h of medium replacement, 10 μL of CCK-8 solution was added to each well. After further incubation at 37°C for 1 h, the absorbance of each well was measured at 450 nm using a microplate reader to calculate cell viability. Results showed that the JNK inhibitor BSJ-04-122 at working concentrations of 15 μM and below had no significant cytotoxicity (see...). Figure 17 (A) in the middle.

[0175] (2) Experiments involving RABV CVS11 strain infection after transient overexpression of TAB2 protein and treatment with JNK inhibitor BSJ-04-122.

[0176] For specific steps, see Example 2. After 1 hour of infection, the medium was replaced with DMEM containing 5% fetal bovine serum with different concentrations of the JNK inhibitor BSJ-04-122. The cell pellet was collected 24 hours after infection and stored at -80°C.

[0177] (3) Western blotting was used to detect the effects of different concentrations of the JNK inhibitor BSJ-04-122 on MAPK signaling pathway-related proteins and RABV proteins after treatment with RABV and infection.

[0178] Western blotting was performed using antibodies Anti-RABV N (prepared and preserved in our laboratory), Anti-JNK1+JNK2+JNK3 (abcam; catalog number ab179461), and Anti-phospho-JNK1+JNK2+JNK3 (abcam; catalog number ab124956) to detect the expression of corresponding MAPK signaling pathway proteins and RABV proteins. Specific steps are detailed in Example 1, and results are shown below. Figure 17 B. The results showed that treatment with the JNK inhibitor BSJ-04-122 significantly inhibited JNK protein phosphorylation, with 10 μM of BSJ-04-122 showing good inhibitory effects.

[0179] (4) Western blotting analysis of the effects of transient TAB2 gene overexpression on MAPK signaling pathway-related proteins and RABV proteins after RABV infection and treatment with the JNK inhibitor BSJ-04-122.

[0180] Western blotting was used to detect the expression of RABV protein and MAPK signaling pathway proteins. Specific steps are described in Example 1, and the results are shown in the figure below. Figure 17 C. Gray-scale analysis showed that TAB2 protein overexpression promoted JNK protein phosphorylation and RABV replication. However, after treatment with the JNK inhibitor BSJ-04-122, the promoting effect of TAB2 protein overexpression on p38 protein phosphorylation and RABV replication remained, indicating that TAB2 protein does not promote RABV replication through the JNK / MAPK signaling pathway (see [link to study].) Figure 17 D and Figure 17 (E in the text)

[0181] Example 13: Effect of TAK1 inhibitor Takinib on TAB2-promoted RABV replication

[0182] (1) Effects of different concentrations of the TAK1 inhibitor Takinib on the viability of N2a cells

[0183] The N2a cell line was counted and set at 1×10⁻⁶. 4Cells were seeded per well in 96-well plates and incubated at 37°C for 20 h. The medium was then replaced with 5% fetal bovine serum DMEM containing different concentrations of the TAK1 inhibitor Takinib (100 μL / well). After 24 h of medium replacement, 10 μL of CCK-8 solution was added to each well. After further incubation at 37°C for 1 h, the absorbance of each well was measured at 450 nm using a microplate reader to calculate cell viability. Results showed that the TAK1 inhibitor Takinib at working concentrations of 30 μM and below had no significant cytotoxicity (see...). Figure 18 (A) in the middle.

[0184] (2) Experiments involving RABV CVS11 strain infection after transient overexpression of TAB2 protein and treatment with the TAK1 inhibitor Takinib.

[0185] For specific steps, see Example 2. After 1 hour of infection, the medium was replaced with DMEM containing 5% fetal bovine serum with different concentrations of the TAK1 inhibitor Takinib. The cell pellet was collected 24 hours after infection and stored at -80°C.

[0186] (3) Western blotting to detect the effects of different concentrations of the TAK1 inhibitor Takinib on MAPK signaling pathway-related proteins and RABV proteins after treatment with RABV and infection.

[0187] Western blotting was used to detect the expression of corresponding MAPK signaling pathway proteins and RABV proteins using the following antibodies: Anti-RABV N (prepared and preserved in our laboratory), p38 MAPK Antibody (Cell Signaling Technology; catalog number 9212), Phospho-p38 MAPK Antibody (Affinity; catalog number AF4001), Anti-JNK1+JNK2+JNK3 (abcam; catalog number ab179461), Anti-phospho-JNK1+JNK2+JNK3 (abcam; catalog number ab124956), Anti-TAK1 (abcam; Cat#ab109526), ​​and Anti-phospho-TAK1 (abcam; Cat#ab109404). Specific steps are detailed in Example 1, and results are shown below. Figure 18 In section B, the results showed that treatment with the TAK1 inhibitor Takinib significantly inhibited the phosphorylation of p38, JNK, and TAK1 proteins, with 15 μM Takinib exhibiting good inhibitory effects (see section B). Figure 18 (B in the middle).

[0188] (4) Western blotting analysis of the effects of transient TAB2 gene overexpression on MAPK signaling pathway-related proteins and RABV proteins after RABV infection with the TAK1 inhibitor Takinib. C DEFG: Western blotting was used to detect the expression of RABV and MAPK signaling pathway proteins. Specific steps are described in Example 1, and the results are shown in the figure below. Figure 18 C. Gray-scale analysis showed that TAB2 protein overexpression promoted the phosphorylation of p38, JNK, and TAK1 proteins and RABV replication. However, treatment with the TAK1 inhibitor Takinib reversed the promoting effects of TAB2 protein overexpression on p38, JNK, and TAK1 protein phosphorylation and RABV replication. This indicates that TAB2 protein promotes RABV replication through the TAK1-p38 / MAPK signaling pathway, and the TAK1 inhibitor Takinib may be a potential drug for treating RABV infection (see [link to article]). Figure 18 D in Figure 18 E in Figure 18 F and Figure 18 (G in the middle).

[0189] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a substance capable of inhibiting the expression of murine TAB2 protein in the preparation of a drug for treating rabies, characterized in that, The mouse-derived TAB2 protein has the NCBI accession number NP_001346463.1; the substance capable of inhibiting the expression of the mouse-derived TAB2 protein is a substance that knocks down the expression of the TAB2 protein; the substance that knocks down the expression of the TAB2 protein is TAB2 siRNA, and the TAB2 siRNA is siRNA1 with the nucleotide sequence shown in SEQ ID NO.9, or siRNA2 with the nucleotide sequence shown in SEQ ID NO.10, or siRNA3 with the nucleotide sequence shown in SEQ ID NO.11, or a mixture of siRNA1, siRNA2 and siRNA3; the rabies is rabies caused by rabies virus of RABV CVS11 strain or RABV SRV9 strain.

2. The application of a substance capable of inhibiting the expression of murine TAB2 protein in the preparation of a drug for inhibiting rabies virus replication, characterized in that, The mouse-derived TAB2 protein has the NCBI accession number NP_001346463.1; the substance capable of inhibiting mouse-derived TAB2 protein expression is a substance that knocks down TAB2 protein expression; the substance that knocks down TAB2 protein expression is TAB2 siRNA, wherein the TAB2 siRNA is siRNA1 with the nucleotide sequence shown in SEQ ID NO.9, or siRNA2 with the nucleotide sequence shown in SEQ ID NO.10, or siRNA3 with the nucleotide sequence shown in SEQ ID NO.11, or a mixture of siRNA1, siRNA2 and siRNA3; the rabies virus is RABV CVS11 strain or RABV SRV9 strain.

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