Application of host factor snapin in development of new rabies vaccine and remedy

Abstract

The application of host factor Snapin in rabies virus new vaccine and treatment drug research belongs to the field of biological medicine technology. In order to study the influence of host factor Snapin on RABV replication and further lay a foundation for the research of rabies virus vaccine and rabies treatment target screening, the present application proves that the host factor Snapin promotes RABV replication as a positive regulation factor by overexpressing Snapin gene and knocking down Snapin gene, and further constructs a recombinant rabies virus rCVS11-Snapin expressing an exogenous protein Snapin, and finds that the recombinant virus has higher in vitro proliferation characteristics compared with the parent strain CVS11, and can be used for rabies virus vaccine preparation. In addition, the present application also finds that knocking down the host factor Snapin can inhibit the replication of rabies virus, and the host factor Snapin can be used as a new target for treating rabies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of the host factor Snapin in the development of novel rabies virus vaccines and treatment drugs. Background Technology

[0002] Rabies is a serious neurotropic zoonotic disease caused by the rabies virus (RABV), and there is currently no effective clinical treatment. Given the lack of effective treatments for rabies, strengthening preventative vaccination to control the spread of the disease and screening for novel rabies therapeutic targets and drugs are of great significance for the comprehensive prevention and clinical treatment of rabies.

[0003] Rabies virus (RABV) is a single-stranded, negative-sense RNA virus belonging to the family Rhabdoviridae and the genus Rabiesvirus. From its 3' to 5' ends, it encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase protein (L). These five structural proteins work together to regulate viral replication, transcription, assembly, and budding. As a neurotropic virus, the key factor in RABV infection is protecting the integrity of axons, dendrites, and synapses. The host protein Snapin is a synaptosome-associated protein, highly conserved throughout biological evolution, and expressed in neurons of the cerebral cortex, hippocampus, cerebellum, hypothalamus, and spinal cord. Studies have shown that... snapin In neurons with gene deletion, late retrograde transport is inhibited, leading to significant accumulation of organelles, reduced neuronal viability, dendritic development defects, and axonal swelling. However, whether the Snapin protein affects RABV replication has not yet been reported. Summary of the Invention

[0004] To investigate the impact of the host factor Snapin on RABV replication and further lay the foundation for research on rabies virus vaccines and rabies therapeutic targets, this invention utilizes overexpression of... Snapin Genes and knockdown Snapin Genetic studies have confirmed that the host factor Snapin acts as a positive regulator promoting RABV replication. Therefore, this invention constructed a recombinant rabies virus rCVS11-Snapin expressing the exogenous protein Snapin, and found that this recombinant virus exhibits higher in vitro replication characteristics compared to the parent strain CVS11, making it suitable for the preparation of rabies virus vaccines. Furthermore, this invention also found that knocking down the host factor Snapin can inhibit rabies virus replication, suggesting that Snapin could serve as a novel therapeutic target for rabies.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention proposes the following technical solution:

[0006] The first objective of this invention is to provide the use of overexpression of the murine host factor Snapin in promoting rabies virus replication, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] A second objective of this invention is to provide a method for constructing recombinant rabies virus rCVS11-Snapin, the method comprising the following steps:

[0008] (1) The artificially optimized Snapin gene sequence was inserted between the G and L genes of the whole genome of rabies virus CVS11 to obtain the recombinant plasmid CVS11-Snapin; the nucleotide sequence of the artificially optimized Snapin gene is shown in SEQ ID NO.1;

[0009] (2) The recombinant plasmid obtained in step (1) was co-transfected with helper plasmids pD-N, pD-P, pD-L and pD-G into BSR cells. After culture, the supernatant was harvested to obtain recombinant rabies virus rCVS11-Snapin.

[0010] In one embodiment of the present invention, step (1) is performed after artificial optimization. Snapin Add to both ends of the gene Bsiw I, Sac The recombinant plasmid CVS11-Snapin was obtained by double digestion of the II restriction site and then inserted into the CVS11 plasmid.

[0011] In one embodiment of the present invention, the specific method of step (1) is to optimize the artificially optimized... Snapin The gene was inserted into the pcDNA3.1 vector to obtain the recombinant expression plasmid pcDNA3.1-Snapin, which was then introduced into the vector. Bsiw I, Sac The recombinant expression plasmid pcDNA3.1-Snapin was amplified using upstream and downstream primers at restriction site II, and the target fragment was obtained by gel extraction; restriction endonucleases were then used to amplify the fragment. Bsiw I and Sac II. The CVS11 vector was double-digested, and the digested products were purified and recovered. The target fragment and the digested products were ligated using a seamless ligase to obtain the recombinant plasmid CVS11-Snapin.

[0012] In one embodiment of the present invention, the ratio of the amounts of auxiliary plasmids pD-N, pD-P, pD-L, and pD-G in step (2) is 10:5:2:3.

[0013] In one embodiment of the present invention, the amounts of auxiliary plasmids pD-N, pD-P, pD-L and pD-G used in step (2) are 0.625 µg, 0.3125 µg, 0.125 µg and 0.1875 µg, respectively.

[0014] A third objective of this invention is to provide a recombinant rabies virus rCVS11-Snapin constructed using the above-described method.

[0015] A fourth objective of this invention is to provide the application of the above-mentioned recombinant rabies virus rCVS11-Snapin in the preparation of rabies virus vaccines.

[0016] A fifth objective of this invention is to provide the application of knocking down the murine host factor Snapin in inhibiting rabies virus replication, the amino acid sequence of which is shown in SEQ ID NO.2.

[0017] A sixth object of the present invention is to provide the use of a substance capable of inhibiting the expression of the murine host factor Snapin in the preparation of a medicament for treating rabies, the amino acid sequence of which is shown in SEQ ID NO.2.

[0018] In one embodiment of the present invention, the substance capable of inhibiting the expression of the mouse host factor Snapin is a substance that knocks down the mouse host factor Snapin.

[0019] In one embodiment of the present invention, the substance that knocks down the mouse host factor Snapin is a mixture of three siRNAs targeting different sites on Snapin mRNA: siSnapin1, siSnapin2, and siSnapin3. The sense strand sequence of siSnapin1 is shown in SEQ ID NO.3, and the antisense strand sequence is shown in SEQ ID NO.4. The sense strand sequence of siSnapin2 is shown in SEQ ID NO.5, and the antisense strand sequence is shown in SEQ ID NO.6. The sense strand sequence of siSnapin3 is shown in SEQ ID NO.7, and the antisense strand sequence is shown in SEQ ID NO.8.

[0020] The beneficial effects of this invention are:

[0021] This invention utilizes overexpression Snapin Genes and knockdown SnapinGenetic studies have confirmed that the host factor Snapin acts as a positive regulator promoting RABV replication. Therefore, this invention constructed a recombinant rabies virus rCVS11-Snapin expressing the exogenous protein Snapin, and found that this recombinant virus exhibits higher in vitro replication characteristics compared to the parent strain CVS11, making it suitable for rabies vaccine preparation. Furthermore, this invention also discovered that knocking down the host factor Snapin can inhibit rabies virus replication, suggesting that Snapin could serve as a novel therapeutic target for rabies. Attached Figure Description

[0022] Figure 1 The image shows the results of agarose gel electrophoresis for identifying the recombinant plasmid pcDNA3.1-Snapin.

[0023] Figure 2 The figure shows the effect of host factor Snapin overexpression on RABV N mRNA transcription levels and RABV N protein levels; among them, Figure 2 Figure A in the graph represents the effect of host factor Snapin overexpression on RABV N mRNA transcription levels. Figure 2 Figure B in the figure shows the effect of host factor Snapin overexpression on RABV N protein levels.

[0024] Figure 3 The figure shows the effect of host factor Snapin overexpression on viral titer in cell supernatant after viral infection.

[0025] Figure 4 The figure shows the effects of host factor Snapin gradient overexpression on RABV N mRNA transcription levels and RABV N protein levels; among them... Figure 4 Figure A shows the effect of host factor Snapin gradient overexpression on RABV N mRNA transcription levels. Figure 4 In the figure, B represents the effect of host factor Snapin gradient overexpression on RABV N protein levels.

[0026] Figure 5 The graph shows the knockdown efficiency of the host factor Snapin.

[0027] Figure 6 Figure showing the effect of Snapin gene knockdown on viral titer in cell supernatant after viral infection;

[0028] Figure 7 The figure shows the effect of Snapin gene knockdown on RABV N mRNA transcription levels and RABV N protein levels; among them... Figure 7Figure A in the diagram shows the effect of Snapin gene knockdown on RABV N mRNA transcription levels. Figure 7 Figure B in the diagram shows the effect of Snapin gene knockdown on RABV N protein levels.

[0029] Figure 8 Diagram illustrating the construction strategy for the recombinant plasmid CVS11-Snapin;

[0030] Figure 9 The image shows the results of identifying the Snapin gene obtained from the gel recovery.

[0031] Figure 10 Figure showing the results of DFA identification of BSR cells infected with recombinant rabies virus rCVS11-Snapin;

[0032] Figure 11 The figure shows the results of genetic stability analysis of recombinant rabies virus rCVS11-Snapin;

[0033] Figure 12 The image shows the results of indirect immunofluorescence identification of N2a cells infected with recombinant rabies virus rCVS11-Snapin.

[0034] Figure 13 Figure showing the results of Western Blot identification of N2a cells infected with recombinant rabies virus rCVS11-Snapin;

[0035] Figure 14 The growth kinetics curves of recombinant rabies virus rCVS11-Snapin on N2a cells are shown; among them, Figure 14 In the figure, A represents the growth kinetics curve of N2a cells seeded with MOI=0.1. Figure 14 B in the figure represents the growth kinetics curve of N2a cells seeded with MOI=1; Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1: The effect of Snapin on RABV replication

[0040] 1. Target sequence synthesis

[0041] According to GenBank's announcement Snapin The gene sequence (NM_133854) was optimized based on the codon preferences of mice, and the optimized sequence was synthesized by Sangon Biotech Co., Ltd. Snapin The gene fragment (see SEQ ID NO.1, and its corresponding amino acid sequence is shown in SEQ ID NO.2) was ligated into the pcDNA3.1 vector to obtain the recombinant plasmid pcDNA3.1-Snapin. The recombinant plasmid was identified by agarose gel electrophoresis, and the identification results are shown in […]. Figure 1 .

[0042] SEQ ID NO.1:

[0043] ATGGCCGCCGCCGGCAGCGCCGCCGTGAGCGGCGCCGGCACCCCCGTGGCCGGCCCTACCGGCAGGGACCTGTTCGCCGAGGGCCTGCTGGAGTTCCTGAGGCCTGCCGTGCAGCAGCTGGACAGCCACGTGCACGCCGTGAGGGAGCCAGGTGGAGCTGAGGGAGCAGATCGACAACCTGGCCACCGAGCTGTGCAGGATCA ACGAGGACCAGAAGGTGGCCCTGGACCTGGACCCTTACGTGAAGAAGCTGTTGAATGCTAGAAGAAGAGTGGTGCTGGTGAATAATATCCTGCAGAATGCTCAGGAGAGACTGAGAAGGCTGAATCATTCTGTGGCTAAGGAAACAGCTAGAAGAAGGGCTATGCTGGATTCTGGAGTGTATCCACCTGGCTCTCCTTCTAAATGA

[0044] SEQ ID NO.2:

[0045] MAAAGSAAVSGAGTPVAGPTGRDLFAEGLLEFLRPAVQQLDSHVHAVRESQVELREQIDNLATELCRINEDQKVALDLDPYVKKLLNARRRVVLVNNILQNAQERLRRLNHSVAKETARRRAMLDSGVYPPGSPSK

[0046] 2. Effects of host factor Snapin overexpression on RABV replication

[0047] Recombinant plasmids pcDNA3.1-Snapin and pcDNA3.1 were transfected into N2a cells (mouse neuroblastoma cells). Twenty-four hours after transfection, RABV CVS-11 strain was inoculated at an MOI of 0.1. Viral titer, RABV N mRNA transcription level, and RABV N protein level in the cell supernatant were measured at 24, 36, and 48 hours post-infection. Results showed that Snapin gene overexpression significantly increased RABV N mRNA transcription level and N protein level at 36 and 48 hours post-infection (see [link to relevant documentation]). Figure 2 Viral titers in post-infection cell supernatants showed an increasing trend (see [link to relevant documentation]). Figure 3 Furthermore, this invention also examined the effects of gradient overexpression of the host factor Snapin on RABV N mRNA transcription levels and RABV N protein levels. The specific procedures were as follows: Each well of a six-well plate was transfected with 0 μg, 0.1 μg, 0.5 μg, 1 μg, and 2 μg of pcDNA3.1-Snapin (quantification per well was 2 μg; any amount less than 2 μg was supplemented with empty pcDNA3.1 vector). The results showed that RABV N mRNA transcription levels and RABV N protein levels increased in a gradient with Snapin expression (see...). Figure 4 ).

[0048] 3. The effect of host factor Snapin knockdown on RABV replication

[0049] To detect the interference efficiency of siRNAs specifically targeting the host protein Snapin, three siRNAs targeting different sites on Snapin mRNA (siSnapin) (as shown in Table 1) were mixed in a 1:1:1 ratio and transfected into N2a cells. A negative control (siNC) without any mRNA targeting was also included. After 36 h of transfection, the relative levels of Snapin mRNA in the cells were detected using RT-qPCR. The results are as follows: Figure 5As shown, the Snapin mRNA level was significantly reduced in N2a cells co-transfected with siSnapin. Three siSnapin lines were transfected into N2a cells at a 1:1:1 ratio (25 pmol per well in a 6-well plate). 24 h post-transfection, RABV CVS11 strain was inoculated at MOI=0.1. Viral titer, RABV N mRNA transcription level, and RABV N protein level in the infected cell supernatant were measured at 24 h, 36 h, and 48 h post-inoculation. The results showed that... Snapin After gene knockdown, the viral titer in the cell supernatant was significantly reduced 48 h after infection (see [link to article]). Figure 6 At 36 h and 48 h post-infection, RABV N mRNA transcription levels and RABV N protein levels were significantly reduced (see [link to relevant documentation]). Figure 7 The results of the above host factor Snapin knockdown experiment indicate that... Snapin Gene knockdown inhibits RABV replication, and the host factor Snapin could serve as a novel target for the treatment of rabies.

[0050] Table 1. siRNA sequence information targeting Snapin

[0051]

[0052] Example 2: Construction and identification of recombinant rabies virus rCVS11-Snapin

[0053] 1. Primer design and synthesis

[0054] Design-specific amplification Snapin Primers for the gene (see Table 2). The upstream primer Snapin-F (with an enzyme cleavage site introduced at the 5' end) was designed based on the reference synthetic sequence SEQ ID NO.1. Bsiw I and upstream homologous sequence of canine virus CVS11 strain), downstream primer Snapin-R (introducing restriction site at 3' end) SacII and the upstream homologous sequence of canine rabies virus CVS11 strain), and primers JD-F and JD-R were designed and identified based on the full-length plasmid sequence of rabies virus CVS11 strain. The full-length plasmid of rabies virus CVS11 strain has been published in the following literature "An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice" doi: 10.1007 / s11262-014-1049-9. Epub 2014 Feb 18.

[0055] Table 2 Primer Information

[0056]

[0057] Note: Double underlines indicate homologous sequences of CVS11 strain, and single underlines indicate restriction enzyme sites.

[0058] 2. Construction and identification of recombinant plasmid CVS11-Snapin

[0059] (1) Target gene Snapin amplification

[0060] The construction strategy of recombinant plasmid CVS11-Snapin is as follows: Figure 8 As shown, the exogenous gene Snapin was inserted between the G and L proteins of the RABVCVS11 strain. Using the constructed pcDNA3.1-Snapin as a template, Snapin-F and Snapin-R were used as primers to amplify the target gene Snapin. The reaction conditions are shown in Table 3. The target fragment was separated and recovered by 1% agarose gel electrophoresis. The fragment size was consistent with the expected size, indicating that the target fragment Snapin was successfully amplified.

[0061] Table 3. PCR reaction procedure for amplifying the target gene Snapin

[0062]

[0063] (2) Ligation and identification of recombinant plasmid CVS11-Snapin

[0064] The enzyme digestion conditions for the CVS11 vector are shown in Table 4.

[0065] Table 4 Enzyme digestion conditions for CVS11 vector

[0066]

[0067] Using restriction endonucleases Bsiw I and Sac II. The CVS11 vector was double-digested. The purified digested product was then ligated with the target fragment recovered from gel excision in step (1) using a seamless ligase at 50°C for 20 min. The ligation product was transformed into HST08 competent cells. Single clones were picked and identified by PCR using primers JD-F and JD-R (reaction conditions are shown in Table 5). The clones were then sent to Sangon Biotech for sequencing. The results showed that the sequence was 100% homologous to the synthesized Snapin gene sequence. Enzyme digestion was also performed, and bands of the size of Snapin were observed after digestion (see Table 5). Figure 9 The above results indicate that the recombinant RABV full-length plasmid CVS11-Snapin was successfully constructed, and the target fragment Snapin was successfully ligated between the GL segments of the RABV CVS11 strain genome.

[0068] Table 5. PCR reaction procedure for identifying the Snapin gene.

[0069]

[0070] 3. The rescue of recombinant rabies virus rCVS11-Snapin

[0071] BSR cells were seeded into 6-well plates and cultured at 37°C with 5% CO2. When the cells reached 80-90% confluency, the successfully constructed full-length plasmid CVS11-Snapin and helper plasmids pD-N, pD-P, pD-M, and pD-G (all of which are disclosed in the prior art literature entitled “Using rabies virus vaccine strain SRV9 as viral vector to express exogenous gene”, DOI: 10.1007 / s11262-014-1160-y.) were co-transfected into BSR cells to rescue the recombinant virus. The specific operating steps are as follows: Take a 1.5 mL EP tube and add 250 μL OPTI-MEM, 8 μL P3000, and the full-length recombinant plasmid CVS11-Snapin and helper plasmids pD-N, pD-P, pD-M, and pD-G (the amounts added are shown in Table 6). Gently mix and let stand at room temperature for 5 min to obtain solution A. Take another 1.5 mL EP tube and add 50 μL OPTI-MEM and 10 μL Lipofectamine™ 3000. Gently mix and let stand at room temperature for 5 min to obtain solution B. Add solution B to solution A, gently mix, and let stand at room temperature for 15 min. During this period, wash the cells once with OPTI-MEM, then add 1.5 mL OPTI-MEM to each well. Then, disperse the mixture dropwise into a six-well plate and culture at 37°C and 5% CO2 for 5 h. After 5 h, replace the medium with DMEM containing 10% fetal bovine serum for further culture. Supernatant was collected at 3, 5, 7, 9, 11 and 13 days post-transfection for RABV DFA detection.

[0072] Table 6. Amount of each plasmid used for transfection per well

[0073]

[0074] 4. Identification of recombinant rabies virus rCVS11-Snapin

[0075] (1) DFA identification of recombinant rabies virus rCVS11-Snapin

[0076] To determine whether the recombinant virus was successfully rescued, the supernatant collected in step 3 was subjected to DFA identification. The specific steps were as follows: BSR cells were passaged normally and seeded into 96-well plates (100 μL / well). Then, 50 μL of the collected supernatant was simultaneously added to the cells in the 96-well plates and incubated at 37°C in a 5% CO2 incubator for 48 h. After 48 h, the supernatant was discarded, and 150 μL of 80% cold acetone (stored at -20°C) was added to each well for fixation at room temperature for 30 min. The fixative was discarded, and the cells were washed three times with PBST. After discarding the liquid, the cells were gently tapped on paper to expel the liquid from the wells. 200-fold diluted FITC-labeled mouse anti-rabies virus N protein antibody (40 μL / well) was added to the wells in the dark and incubated at 37°C for 1 h. After 1 h, the antibody was discarded, and the cells were washed three times with PBST. The 96-well plates were observed under a fluorescence microscope. The results are shown in the figure (see figure). Figure 10 Compared with normal cells, BSR cells infected with recombinant rabies virus rCVS11-Snapin showed obvious green fluorescence under a fluorescence microscope, indicating that the recombinant rabies virus rCVS11-Snapin was initially successfully rescued.

[0077] (2) Genetic stability of recombinant rCVS11-Snapin

[0078] RNA was extracted from the P1, P3, P6, and P9 generations of the recombinant virus rCVS11-Snapin and reverse transcribed into cDNA. Using the cDNA as a template and JD-F and JD-R as identification primers, PCR amplification was performed. Agarose gel electrophoresis results showed that Snapin was stably expressed during viral replication (see...). Figure 11 ).

[0079] (3) Indirect immunofluorescence identification

[0080] N2a cells were passaged normally and seeded into 96-well plates. The maternal virus CVS11 and recombinant virus rCVS11-Snapin were simultaneously inoculated. After culturing at 37°C and 5% CO2 for 48 h, the supernatant was discarded. 150 μL of 80% cold acetone was added to each well, and the plates were fixed at room temperature for 30 min. The fixative was discarded, and 150 μL of PBST was added per well. The cells were washed three times with PBST, and the liquid in the wells was gently tapped on paper on the last wash. 50 μL of anti-Snapin protein antibody (1:100 dilution) was added to each well of the CVS11 and rCVS11-Snapin groups, respectively. The cells were incubated at 37°C for 1 h, followed by three washes with PBST. Evans blue staining solution (1:500 dilution) and FITC-labeled goat anti-mouse secondary antibody (1:500 dilution) were then added. The plates were incubated at 37°C in the dark for 1 h, followed by three washes with PBST. The results were observed under a fluorescence microscope in the 96-well plates. The results are as follows: Figure 12As shown, compared with the negative control group of normal N2a cells and N2a cells infected with the parent virus CVS11 strain, N2a cells infected with recombinant rabies virus rCVS11-Snapin showed obvious green fluorescence under a fluorescence microscope, indicating that recombinant rabies virus rCVS11-Snapin can successfully express the exogenous protein Snapin.

[0081] (4) Western Blot identification

[0082] Supernatants from N2a cells infected with the parent virus CVS11 and recombinant virus rCVS11-Snapin, respectively, were mixed with SDS-PAGE protein loading buffer (5X) and boiled in water for 6 min. SDS-PAGE electrophoresis was used to separate proteins from the prepared samples, which were then transferred to nitrocellulose membranes (NC). The NC membranes were blocked with PBST containing 5% skim milk powder at room temperature for 2 h to block non-specific protein binding sites. After blocking, Snapin polyclonal antibody serum obtained through immunization was used as the primary antibody (dilution: 1:500), and incubated overnight at 4°C. The cells were washed 5 times with PBST, 5 min each time. HRP-labeled goat anti-mouse IgG diluted with blocking buffer was used as the secondary antibody (1:10000), and incubated at room temperature for 1 h. After incubation, the cells were washed 5 times with PBST, 5 min each time. The results were analyzed using a gel imaging system. The results are as follows: Figure 13 As shown, the recombinant virus rCVS11-Snapin showed the target band at around 18 Kd, which is consistent with the size of the target fragment of Snapin plus RABV G and L genes.

[0083] Example 3: Determination of growth kinetics of recombinant rabies virus rCVS11-Snapin

[0084] To determine the growth kinetics of the parent virus and recombinant rabies virus in N2a cells, N2a cells were normally passaged into T25 cell flasks. When the cells reached 80-90% confluence, they were infected with the parent virus CVS11 and the recombinant virus rCVS11-Snapin at MOI=0.1 and MOI=1, respectively. The cells were then incubated at 37℃ in a 5% CO2 incubator. Supernatants were collected at 24 h, 48 h, 72 h, and 96 h post-infection for virus titer determination, and virus growth kinetics curves were plotted. Results are as follows: Figure 14 As shown, the viral titer of recombinant virus rCVS11-Snapin was significantly higher than that of the parent strain CVS11 at MOI=1, indicating that recombinant viruses expressing Snapin protein have a higher level of intracellular replication.

[0085] 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. A method for constructing recombinant rabies virus rCVS11-Snapin, characterized in that, Includes the following steps: (1) The artificially optimized Snapin gene sequence was inserted between the G and L genes of the whole genome of rabies virus CVS11 to obtain the recombinant plasmid CVS11-Snapin; the nucleotide sequence of the artificially optimized Snapin gene is shown in SEQ ID NO.1; (2) The recombinant plasmid obtained in step (1) was co-transfected with helper plasmids pD-N, pD-P, pD-L and pD-G into BSR cells. After culture, the supernatant was harvested to obtain recombinant rabies virus rCVS11-Snapin.

2. The construction method according to claim 1, characterized in that, The specific method of step (1) is to insert the artificially optimized Snapin gene into the pcDNA3.1 vector to obtain the recombinant expression plasmid pcDNA3.1-Snapin, and then use the introduced... Bsiw I, Sac The recombinant expression plasmid pcDNA3.1-Snapin was amplified using upstream and downstream primers at restriction site II, and the target fragment was obtained by gel extraction; restriction endonuclease was then used to amplify the fragment. Bsiw I and Sac II. The full-length plasmid of rabies virus strain CVS11 was double-digested with enzymes, and the digested products were purified and recovered. The target fragment and the digested products were ligated using a seamless ligase to obtain the recombinant plasmid rCVS11-Snapin.

3. The construction method according to claim 1, characterized in that, In step (2), the ratio of the amounts of helper plasmids pD-N, pD-P, pD-L, and pD-G is 10:5:2:

3.

4. The recombinant rabies virus rCVS11-Snapin obtained by the construction method according to any one of claims 1-3.

5. The application of a substance capable of inhibiting the expression of the murine host factor Snapin in the preparation of a drug for treating rabies, characterized in that, The amino acid sequence of the murine host factor Snapin is shown in SEQ ID NO.2; the substance that can inhibit the expression of murine host factor Snapin is a substance that knocks down murine host factor Snapin, and the substance that knocks down murine host factor Snapin is a mixture of three siRNAs targeting different sites on Snapin mRNA: siSnapin1, siSnapin2, and siSnapin3. The sense strand sequence of siSnapin1 is shown in SEQ ID NO.3, and the antisense strand sequence is shown in SEQ ID NO.4; the sense strand sequence of siSnapin2 is shown in SEQ ID NO.5, and the antisense strand sequence is shown in SEQ ID NO.6; the sense strand sequence of siSnapin3 is shown in SEQ ID NO.7, and the antisense strand sequence is shown in SEQ ID NO.8.

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