Recombinant rabies virus genome plasmid, recombinant rabies virus and preparation method and application thereof

Abstract

The invention discloses a recombinant rabies virus genome plasmid, a recombinant rabies virus and a preparation method and application thereof, and the preparation method specifically comprises the following steps: (1) introducing a sequence 1 and a sequence 2 into the recombinant rabies virus genome plasmid, so that the recombinant rabies virus genome plasmid has more selectivity in modification and application; (2) introducing a fluorescent protein gene and a functional probe into the recombinant rabies virus genome plasmid, so that the recombinant rabies virus genome plasmid has the functions of expressing the fluorescent protein and the functional probe; (3) preparing a corresponding recombinant rabies virus which can express fluorescent protein with high sensitivity for cell marking and can also effectively express a functional probe for functional research; the method has wide application value and market prospect in the aspects of neural circuit structure and function analysis, neuron sparse marking, vaccine and drug research and development, disease model establishment, neural network loss and reconstruction detection, virus replication or pathogenic mechanism research and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant rabies virus genome plasmid, a recombinant rabies virus, and a preparation method and application thereof. Background Art

[0002] Rabies virus is a single-stranded, non-segmented negative-strand RNA virus. Its genome consists of 7 functional regions, including a 3′ leader region, 5 structural genes (N, P, M, G and L proteins, etc.) and a 5′ non-transcribed region. Each structural gene contains a different transcription start signal, and there are non-transcribed spacers of different sizes and sequences between the structural genes. Retrograde tracing tool viruses based on CVS-N2c-ΔG recombinant rabies virus can be used to analyze the structure and function of neural circuits. They are widely used in the field of neuroscience and have great economic value. However, the existing CVS-N2c-ΔG still has problems such as limited modification sites, low fluorescence expression efficiency, and difficulty in balancing structural and functional studies. Therefore, the development of a recombinant rabies virus with multiple modification sites, highly sensitive expression of fluorescent proteins, and effective expression of functional probes can shorten the experimental cycle, reduce research costs, and improve usage, which is of great significance. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a recombinant rabies virus genome plasmid, a recombinant rabies virus, and a preparation method and application thereof, aiming to solve the problems of limited modification sites of the existing recombinant rabies virus genome plasmid, low efficiency of recombinant rabies virus expression of fluorescence, and difficulty in balancing structural and functional research.

[0004] The technical solution of the present invention is as follows:

[0005] In a first aspect, a method for preparing a recombinant rabies virus genome plasmid is provided, the preparation method comprising:

[0006] A nucleotide sequence 1 as shown in SEQ ID NO.1 was synthesized, and the sequence 1 and CVS-N2c-ΔG-tdTomato plasmid were double-digested with BstZ17I and NheI respectively and then ligated to obtain CVS001 plasmid;

[0007] A nucleotide sequence 2 as shown in SEQ ID NO. 2 was synthesized, and the sequence 2 and the CVS001 plasmid were double-digested with NcoI and AvrII respectively and then connected to obtain the CVS002 plasmid.

[0008] The preferred technical solution, after obtaining the CVS002 plasmid, further comprises:

[0009] Synthesize or amplify a fluorescent protein gene, and connect the fluorescent protein gene and CVS002 plasmid after double digestion with HpaI and SbfI to obtain CVS003 plasmid;

[0010] The functional probe gene is synthesized or amplified, and the functional probe gene and the CVS003 plasmid are double-digested with BsiWI and NheI respectively and then connected to obtain a recombinant rabies virus genome plasmid.

[0011] In a preferred technical solution, the fluorescent protein gene is the mScarlet-I gene; the nucleotide sequence of the mScarlet-I gene is shown in SEQ ID NO.3.

[0012] In a preferred technical solution, the functional probe gene is the SV40 NLS-Cre gene or the SV40 NLS-Flpo gene; the nucleotide sequence of the SV40 NLS-Cre gene is shown in SEQ ID NO.4, and the nucleotide sequence of the SV40 NLS-Flpo gene is shown in SEQ ID NO.5.

[0013] In a second aspect, a recombinant rabies virus genome plasmid is provided. The recombinant rabies virus genome plasmid is prepared using the preparation method described in the first aspect.

[0014] In a third aspect, a recombinant rabies virus is provided, wherein the recombinant rabies virus is obtained by co-transfecting cells with the recombinant rabies virus genome plasmid and the backbone plasmid as described in the second aspect.

[0015] In a fourth aspect, a method for preparing the recombinant rabies virus as described in the fourth aspect is provided, wherein the method comprises:

[0016] The recombinant rabies virus genome plasmid and the backbone plasmid as described in the second aspect are co-transfected into B7GG cells for virus rescue and amplification to obtain the recombinant rabies virus.

[0017] In a preferred technical solution, the preparation method further comprises:

[0018] The recombinant rabies virus is used to infect BHK-N2cG cells to obtain the recombinant rabies virus encapsulated by N2cG.

[0019] In a preferred technical solution, the backbone plasmid includes pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G.

[0020] According to a preferred technical solution, the mass ratio of the recombinant rabies virus genome plasmid, pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G is 4:2:1:1:1.

[0021] In a fifth aspect, there is provided the use of the recombinant rabies virus genome plasmid as described in the second aspect or the recombinant rabies virus as described in the third aspect in neural circuit tracing.

[0022] Beneficial effects: The present invention introduces sequence 1 and sequence 2 into the existing CVS-N2c-ΔG-tdTomato plasmid to establish a new recombinant rabies virus genome plasmid CVS002, which has more selectivity in transformation and application; further, the present invention inserts a fluorescent protein gene and a functional probe gene into the G gene site of the recombinant rabies virus genome plasmid CVS002, respectively, so that it has the functions of expressing fluorescent protein and expressing functional probes, and obtains recombinant rabies virus genome plasmids CVS004 and CVS005. At the same time, the present invention prepares the corresponding recombinant rabies virus, which can not only express fluorescent protein with high sensitivity for cell labeling, but also effectively express functional probes for functional research, and has broad application value and market prospects in neural circuit structure and function analysis, neuron sparse labeling, vaccine and drug development, disease model establishment, detection of neural network loss and reconstruction, virus replication or pathogenic mechanism research, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the map of CVS004 plasmid.

[0024] Figure 2 It is a schematic diagram of the map of CVS005 plasmid.

[0025] Figure 3 This is a comparison of the fluorescence expression efficiency of N2cG-encapsulated CVS004, CVS005 and CVS-N2c-ΔG-tdTomato viruses after infecting HEK-293T cells.

[0026] Figure 4 This is a diagram showing the functional verification results of the CVS004 virus encapsulated by N2cG.

[0027] Figure 5 This is a diagram showing the functional verification results of the CVS005 virus encapsulated by N2cG. DETAILED DESCRIPTION

[0028] The present invention provides a recombinant rabies virus genome plasmid, a recombinant rabies virus, and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0029] The embodiment of the present invention provides a method for preparing a recombinant rabies virus genome plasmid, the preparation method comprising:

[0030] A nucleotide sequence 1 as shown in SEQ ID NO.1 was synthesized, and the sequence 1 and CVS-N2c-ΔG-tdTomato plasmid were double-digested with BstZ17I and NheI respectively and then ligated to obtain CVS001 plasmid;

[0031] A nucleotide sequence 2 as shown in SEQ ID NO. 2 was synthesized, and the sequence 2 and the CVS001 plasmid were double-digested with NcoI and AvrII respectively and then connected to obtain the CVS002 plasmid.

[0032] Specifically, in this example, sequence 1 (as shown in SEQ ID NO.1) with a termination signal (tgaaaaaaa), a gene spacer (ctatt) and a transcription start signal (aacatccct) of the G gene was synthesized, and then double-digested with BstZ17I and NheI and connected and inserted into the CVS-N2c-ΔG-tdTomato plasmid (addgene, catalog number: #73462) that had been double-digested to obtain the CVS001 plasmid; then sequence 2 (as shown in SEQ ID NO.2) with a transcription start signal (aacacccct) of the N gene, a termination signal (tgaaaaaaa), a gene spacer (ctatt) and a transcription start signal (aacatccct) of the G gene was synthesized, and then double-digested with NcoI and AvrII and connected and inserted into the CVS001 plasmid that had been double-digested to obtain a new recombinant rabies virus genome plasmid CVS002. In this example, sequence 1 and sequence 2 are introduced into the CVS-N2c-ΔG-tdTomato plasmid, the transcription start signal corresponding to the N gene of the CVS-N2c-ΔG-tdTomato plasmid is replaced with the transcription start signal of the G gene, and the N gene transcription start signal is introduced to drive the transcription of the exogenous gene, so that the final CVS002 plasmid has multiple modification sites and has more selectivity in modification and application.

[0033] The preferred technical solution, after obtaining the CVS002 plasmid, further comprises:

[0034] Synthesize or amplify a fluorescent protein gene, and connect the fluorescent protein gene and CVS002 plasmid after double digestion with HpaI and SbfI to obtain CVS003 plasmid;

[0035] The functional probe gene is synthesized or amplified, and the functional probe gene and the CVS003 plasmid are double-digested with BsiWI and NheI respectively and then connected to obtain a recombinant rabies virus genome plasmid.

[0036] Specifically, based on the CVS002 plasmid, this embodiment further inserts a fluorescent protein gene and a functional probe gene into the G gene site, so that the recombinant rabies virus genome plasmid finally prepared can express the fluorescent protein and the functional probe with high sensitivity, thereby enabling both structural and functional research.

[0037] In one embodiment, the fluorescent protein gene is the mScarlet-I gene; the nucleotide sequence of the mScarlet-I gene is shown in SEQ ID NO.3.

[0038] In one embodiment, the functional probe gene is the SV40 NLS-Cre gene or the SV40 NLS-Flpo gene; the nucleotide sequence of the SV40 NLS-Cre gene is shown in SEQ ID NO.4, and the nucleotide sequence of the SV40 NLS-Flpo gene is shown in SEQ ID NO.5.

[0039] The embodiment of the present invention provides a recombinant rabies virus genome plasmid, and the recombinant rabies virus genome plasmid is prepared by the preparation method described above.

[0040] The embodiment of the present invention provides a recombinant rabies virus, wherein the recombinant rabies virus is obtained by co-transfecting cells with the recombinant rabies virus genome plasmid and the backbone plasmid as described above.

[0041] The present invention provides a method for preparing the recombinant rabies virus as described above, the method comprising:

[0042] The recombinant rabies virus genome plasmid and backbone plasmid as described above were co-transfected into B7GG cells for virus rescue and amplification to obtain recombinant rabies virus.

[0043] In one embodiment, the preparation method further comprises:

[0044] The recombinant rabies virus is used to infect BHK-N2cG cells to obtain the recombinant rabies virus encapsulated by N2cG.

[0045] In one embodiment, the backbone plasmid includes pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G.

[0046] In one embodiment, the mass ratio of the recombinant rabies virus genome plasmid, pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G is 4:2:1:1:1.

[0047] The embodiments of the present invention provide the use of the above-mentioned recombinant rabies virus genome plasmid or the above-mentioned recombinant rabies virus in neural circuit tracing.

[0048] An embodiment of the present invention provides a use of the above-mentioned recombinant rabies virus genome plasmid or the above-mentioned recombinant rabies virus in neural circuit tracing.

[0049] In one embodiment, the recombinant rabies virus genome plasmid or the recombinant rabies virus is used to prepare a drug for neural circuit tracing.

[0050] In one embodiment, the recombinant rabies virus genome plasmid or the recombinant rabies virus is applied to the analysis of neural circuit structure and function, sparse neuron labeling, vaccine and drug development, disease model establishment, detection of neural network loss and reconstruction, and study of viral replication or pathogenic mechanism.

[0051] The present invention will be further described below by means of specific examples.

[0052] Example 1

[0053] This embodiment provides a recombinant rabies virus genome plasmid and a corresponding recombinant rabies virus, and the preparation steps are as follows:

[0054] 1. Construction of recombinant rabies virus genome plasmid

[0055] Sequence 1 (as shown in SEQ ID NO. 1) with a termination signal (tgaaaaaaa), a gene spacer (ctatt) and a transcription start signal (aacatccct) of the G gene was synthesized, and after double digestion with BstZ17I and NheI, the sequence was connected and inserted into the CVS-N2c-ΔG-tdTomato plasmid (addgene, catalog number: #73462) that had been double digested with the same enzymes. The obtained rabies virus genome plasmid was named CVS001 plasmid; Sequence 2 (as shown in SEQ ID NO. 1) with a transcription start signal (aacacccct) of the N gene, a termination signal (tgaaaaaaa), a gene spacer (ctatt) and a transcription start signal (aacatccct) of the G gene was synthesized. The obtained rabies virus genome plasmid was named CVS002 plasmid after double digestion with NcoI and AvrII and then connected and inserted into the CVS001 plasmid which had been double digested with the same enzymes. The transcription start signal corresponding to the N gene in this genome was replaced by the transcription start signal of the G gene, and the transcription start signal of the N gene of the parent was used to drive the transcription of the exogenous gene. Using pAAV-CAG-mScarlet-I-WPRE-hGH polyA (donated by Brinkase (Shenzhen) Biotechnology Co., Ltd.) as a template, the mScarlet-I gene (as shown in SEQ ID NO.3) was amplified, and after double digestion with HpaI and SbfI, it was inserted into the CVS002 plasmid that had been double digested with the same enzymes. The obtained rabies virus genome plasmid was named CVS003 plasmid; using pAAV-hSyn-SV40 NLS-Cre-WPRE-hGH polyA (donated by Brinkase (Shenzhen) Biotechnology Co., Ltd.) as a template, the SV40 NLS-Cre gene (as shown in SEQ ID NO.4) was amplified, and after double digestion with BsiWI and NheI, it was inserted into the CVS003 plasmid that had been double digested with the same enzymes. The obtained rabies virus genome plasmid was named CVS004 plasmid; the sequence of CVS004 plasmid is shown in SEQ ID NO.6, and the map is shown in Figure 1 As shown; pAAV-hSyn-SV40 NLS-Flpo-WPRE-hGH polyA (donated by Brinkasse (Shenzhen) Biotechnology Co., Ltd.) was used as a template to amplify the SV40 NLS-Flpo gene (as shown in SEQ ID NO.5), which was then double-digested with BsiWI and NheI and inserted into the CVS003 plasmid that had been double-digested with the same enzymes. The obtained rabies virus genome plasmid was named CVS005 plasmid; the map of CVS005 plasmid is shown in Figure 2 All PCR primers, gene synthesis and sequencing in the present invention were completed by Beijing Qingke Biotechnology Co., Ltd.

[0056] 2. Preparation and application of recombinant rabies virus

[0057] B7GG cells were cultured in DMEM medium containing 10% FBS and 1% double antibody (provided by SALK Institute for Biological Studies, USA). Before virus rescue, 12-well plates were plated with a cell density of 40%-50%. 1 mL of DMEM medium containing 10% FBS and no double antibody was added to each well and cultured overnight in a 37°C and 5% (v / v) CO2 incubator. After 16 hours, the medium in the well was removed and 1 mL of reduced serum medium Opti-MEM (Gibco) was added, and the cells were cultured in a 37°C and 5% CO2 incubator for use. The CVS004 and CVS005 plasmids prepared in step 1 were respectively mixed with the backbone plasmids pCAG-B19P (addgene, catalog number: #59925), pCAG-B19N (addgene, catalog number: #59924), pCAG-B19L (addgene, catalog number: #59922), and pCAG-B19G (addgene, catalog number: #59921) at a mass ratio of 4:2:1:1:1, and a total of 9 μg was added to a 1.5 mL centrifuge tube pre-filled with 200 μL of Opti-MEM culture medium, and vortexed on a vortex shaker. After mixing, 18 μL of FUGENE 6 transfection reagent was added, vortexed, and allowed to stand at room temperature for 15 minutes. Then, the plates were added to the corresponding wells of the 12-well plate, and the plates were placed in an incubator at 35°C and 3% (v / v) CO2 for 6 hours. The culture medium in the wells was replaced with a 2% The DMEM medium containing FBS was placed in an incubator at 35°C and 3% CO2 for further culture. After 72-120 hours of transfection, the cells were observed under an inverted fluorescence microscope. The appearance of red fluorescence signals indicated that the CVS004 and CVS005 viruses were successfully rescued.

[0058] The cells were then passaged and amplified for 10-14 days under culture conditions of 35°C and 3% (v / v) CO2 to obtain B19G-encapsulated CVS004 and CVS005 viruses. The supernatants of B19G-encapsulated CVS004 and CVS005 viruses were added to BHK-N2cG cells, respectively, and amplified for 5-7 days to obtain N2cG-encapsulated CVS004 and CVS005 viruses. For details on the steps of virus amplification, concentration, purification and preservation, please refer to the literature (Neural Regen Res. 2023; 18(8): 1827-1833). The titers of the N2cG-encapsulated CVS004 and CVS005 viruses were finally obtained, respectively. 8 IU / mL, 5×10 8 IU / mL. The establishment of the BHK-N2cG stable cell line refers to the patent previously applied for by the applicant (patent application number: CN202411387161.0).

[0059] In order to verify that the efficiency of expressing fluorescent proteins under the condition of expressing functional probes in the present invention is significantly better than that of the existing system, equal amounts (0.01 μL each) of N2cG-encapsulated CVS004, CVS005 and CVS-N2c-ΔG-tdTomato viruses (Neural Regen Res. 2023; 18(8): 1827-1833) were taken to infect HEK-293T cells (CVS004 and CVS005 viruses were experimental groups, and CVS-N2c-ΔG-tdTomato virus was a control group, and the titer was diluted to 2×10 8 IU / mL), and then placed in a 37°C and 5% (v / v) CO2 incubator for further culture, and then fluorescence imaging was performed using the same exposure parameters. Figure 3 As shown: The efficiency of expressing fluorescent protein by CVS004 and CVS005 prepared by the present invention is significantly higher than that of CVS-N2c-ΔG-tdTomato (Neural Regen Res. 2023; 18(8): 1827-1833).

[0060] In order to verify whether CVS004 can express enough Cre to achieve splicing and recombination function, N2cG-encapsulated CVS004 virus was used to infect HEK-293T cells plated in 24-well plates. Three days later, 2 μg of Cre-dependent expression pAAV-CMV-DIO-EGFP-WPRE-hGH polyA plasmid (donated by Brinkase (Shenzhen) Biotechnology Co., Ltd.) was transfected. Non-transfection of pAAV-CMV-DIO-EGFP-WPRE-hGH polyA plasmid was used as a control. Fluorescence imaging was performed one day later. The results are shown in Figure 2. Figure 4 As shown: CVS004 prepared by the present invention can effectively express Cre for cutting DIO elements, so that the inverted green fluorescent protein can be normally flipped and effectively expressed. Similarly, in order to verify whether CVS005 can express enough Flpo to achieve the cutting and recombination function, CVS005 was used to infect HEK-293T cells plated in 24-well plates, and 2 μg of Flpo-dependent expression pAAV-CMV-FDIO-EGFP-WPRE-hGH polyA (donated by Brinkase (Shenzhen) Biotechnology Co., Ltd.) plasmid was transfected 3 days later. The pAAV-CMV-FDIO-EGFP-WPRE-hGH polyA plasmid was not transfected as a control, and fluorescence imaging was performed 1 day later. The results are shown in FIG. Figure 5 As shown: CVS005 prepared by the present invention can effectively express Flpo for cutting FDIO elements, so that the inverted green fluorescent protein can be normally flipped and effectively expressed.

[0061] In summary, the present invention provides a recombinant rabies virus genome plasmid, a recombinant rabies virus, and a preparation method and application thereof, which specifically include: (1) introducing a transcription start and stop signal in front of the N gene, and replacing the transcription start signal of the N gene with the transcription start signal of the G gene, constructing a new rabies virus genome plasmid CVS002, inserting a fluorescent probe to obtain a rabies virus genome plasmid CVS003, inserting functional probes such as Cre and Flpo recombinase at the G gene site, respectively, obtaining rabies virus genome plasmids CVS004 and CVS005, and preparing the corresponding recombinant rabies virus; (2) The recombinant rabies virus prepared by the platform can not only express fluorescent protein with high sensitivity for cell labeling, but also effectively express functional probes for functional research. The present invention solves the problems of limited modification sites, low fluorescence expression efficiency, and difficulty in balancing structural and functional applications of existing methods, and has broad application value and market prospects in neural circuit structure and function analysis, sparse neuron labeling, vaccine and drug development, disease model establishment, detection of neural network loss and reconstruction, virus replication or pathogenic mechanism research, etc.

[0062] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a recombinant rabies virus genome plasmid, characterized in that: The preparation method comprises: A nucleotide sequence 1 as shown in SEQ ID NO.1 was synthesized, and the sequence 1 and CVS-N2c-ΔG-tdTomato plasmid were double-digested with BstZ17I and NheI respectively and then ligated to obtain CVS001 plasmid; A nucleotide sequence 2 as shown in SEQ ID NO. 2 was synthesized, and the sequence 2 and the CVS001 plasmid were double-digested with NcoI and AvrII respectively and then connected to obtain the CVS002 plasmid.

2. The method for preparing the recombinant rabies virus genome plasmid according to claim 1, characterized in that: After obtaining the CVS002 plasmid, it also includes: Synthesize or amplify a fluorescent protein gene, and connect the fluorescent protein gene and CVS002 plasmid after double digestion with HpaI and SbfI to obtain CVS003 plasmid; The functional probe gene is synthesized or amplified, and the functional probe gene and the CVS003 plasmid are double-digested with BsiWI and NheI respectively and then connected to obtain a recombinant rabies virus genome plasmid.

3. The method for preparing the recombinant rabies virus genome plasmid according to claim 2, characterized in that: The fluorescent protein gene is the mScarlet-I gene; the nucleotide sequence of the mScarlet-I gene is shown in SEQ ID NO.

3.

4. The method for preparing the recombinant rabies virus genome plasmid according to claim 2, characterized in that: The functional probe gene is the SV40 NLS-Cre gene or the SV40 NLS-Flpo gene; the nucleotide sequence of the SV40 NLS-Cre gene is shown in SEQ ID NO.4, and the nucleotide sequence of the SV40 NLS-Flpo gene is shown in SEQ ID NO.

5.

5. A recombinant rabies virus genome plasmid, characterized in that: The recombinant rabies virus genome plasmid is prepared by the preparation method according to any one of claims 1 to 4.

6. A recombinant rabies virus, characterized in that: The recombinant rabies virus is obtained by co-transfecting cells with the recombinant rabies virus genome plasmid and the backbone plasmid as claimed in claim 5.

7. The method for preparing the recombinant rabies virus according to claim 6, characterized in that: The preparation method comprises: The recombinant rabies virus genome plasmid and the backbone plasmid as claimed in claim 5 are co-transfected into B7GG cells for virus rescue and amplification to obtain the recombinant rabies virus.

8. The method for preparing the recombinant rabies virus according to claim 7, characterized in that: The preparation method further comprises: The recombinant rabies virus is used to infect BHK-N2cG cells to obtain the recombinant rabies virus encapsulated by N2cG.

9. The method for preparing the recombinant rabies virus according to claim 7, characterized in that: The backbone plasmids include pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G.

10. Use of the recombinant rabies virus genome plasmid as claimed in claim 5 or the recombinant rabies virus as claimed in claim 6 in neural circuit tracing.

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