Recombinant pseudorabies virus for highly-sensitive expression of red fluorescent protein, and preparation method therefor and use thereof

Abstract

Provided are a recombinant pseudorabies virus for highly-sensitive expression of a red fluorescent protein, and a preparation therefor and a use thereof, specifically comprising: (1) constructing a recombinant plasmid to prepare the recombinant pseudorabies virus for highly-sensitive expression of the red fluorescent protein; and (2) a use in neural circuit tracing, wherein the recombinant pseudorabies virus for highly-sensitive expression of the red fluorescent protein prepared by a platform can achieve efficient retrograde trans-synaptic labeling of a neural network. The recombinant pseudorabies virus for highly-sensitive expression of the red fluorescent protein and for retrograde trans-synaptic labeling is successfully obtained, such that the problem of low red fluorescence expression efficiency in existing methods is solved, and the recombinant pseudorabies virus achieves high application value and wide market prospects in neural circuit structure and function analysis, vaccine and drug research and development, disease model establishment, detection of neural network loss and reconstruction, virus replication or pathogenic mechanism research, etc.

Description

A recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity, and its preparation method and application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity, and a preparation method and application thereof. Background Art

[0002] Neural circuits are extremely complex, and abnormalities often lead to brain diseases such as Alzheimer's and Parkinson's. Currently, effective treatments are lacking, primarily due to a lack of understanding of the mechanisms of neural network mutation. Retrograde transsynaptic tracing viruses can be used to analyze the connectivity and function of neural networks between central and peripheral neurons, and have significant application value in the field of neuroscience.

[0003] Pseudorabies virus (PRV) belongs to the herpesvirinae subfamily, with a diameter of approximately 200-250 nm and a complex genome and structure of approximately 150 kb. It is an enveloped, spherical, double-stranded DNA virus. PRV primarily includes the Becker and Bartha strains. The Becker strain is less commonly used in neural circuit research due to its high toxicity and non-specific transmission direction. The Bartha strain, on the other hand, has low cytotoxicity and can only spread retrogradely across multiple synapses, particularly from the periphery to the central nervous system. This has attracted interest among researchers studying the connections between central nervous systems and between the periphery and the central nervous system, and has been widely used in the study of neural circuit structure and function.

[0004] However, the existing PRV-Bartha still has problems such as low efficiency in expressing red fluorescent protein. Therefore, it is of great significance to develop a recombinant pseudorabies virus that can express red fluorescent protein with high sensitivity.

[0005] Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity, and a preparation method and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a recombinant pseudorabies virus that highly sensitively expresses a red fluorescent protein. The recombinant pseudorabies virus that highly sensitively expresses a red fluorescent protein is packaged by a plasmid containing homology arms and efficiently expressing the red fluorescent protein; wherein the plasmid containing homology arms and efficiently expressing the red fluorescent protein is a pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid.

[0009] The second aspect of the present invention provides a method for preparing a recombinant pseudorabies virus that expresses a red fluorescent protein with high sensitivity, characterized in that the method comprises the following steps: 1) using the pCAG-EGFP-CW3SL plasmid as a vector, inserting the left homology arm into the upstream of the pCAG-EGFP-CW3SL plasmid and the right homology arm into the downstream of the pCAG-EGFP-CW3SL plasmid to obtain the pLHA-CAG-mScarletI-T2A-mScarletI-P2 A-mScarletI-CW3SL-RHA plasmid; wherein the left homology arm is denoted as LHA and the right homology arm is denoted as RHA; 2) transfecting the pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid into BHK-21 cells, which are then infected with PRV-Bartha virus, and the cell culture supernatant is collected; after purification, a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity is obtained.

[0010] Furthermore, the step 1) specifically includes: gene synthesis of LHA sequence and RHA sequence, and inserting the LHA sequence into the upstream of the pCAG-EGFP-CW3SL plasmid that has been double-digested with EcoRI and MluI to obtain pLHA-CAG-EGFP-CW3SL plasmid; and then inserting the RHA sequence into the downstream of the pLHA-CAG-EGFP-CW3SL plasmid that has been double-digested with SpeI and HindIII to obtain pLHA-CAG-EGFP-CW3SL-RHA plasmid. gene synthesis of the mScarletI-T2A-mScarletI-P2A-mScarletI sequence, and the mScarletI-T2A-mScarletI-P2A-mScarletI sequence was double-digested with AgeI and EcoRV and then ligated and inserted into the pLHA-CAG-EGFP-CW3SL-RHA plasmid that had been double-digested with the same enzymes to obtain the pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid.

[0011] Furthermore, the nucleotide sequence of the LHA sequence is shown in SEQ ID NO: 1; the nucleotide sequence of the RHA sequence is shown in SEQ ID NO: 2.

[0012] Furthermore, the nucleotide sequence of the mScarletI-T2A-mScarletI-P2A-mScarletI sequence is shown in SEQ ID NO: 3.

[0013] Furthermore, the step 2) specifically includes: using a liposome transfection method to transfect the pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid into BHK-21 cells, replacing the culture medium with fresh complete medium containing 2% fetal bovine serum after 5 hours, and adding PRV-Bartha virus at the same time, collecting the supernatant after 48 hours, diluting and infecting BHK-21 cells, aspirating the supernatant after 1 hour and adding complete medium containing agar, performing plaque picking purification after 48 hours, picking red fluorescent plaques and transferring them to BHK-21 cells in fresh complete medium containing 2% fetal bovine serum, collecting the supernatant after 48 hours, continuing to dilute, infect and plaque picking purification, and repeating 4 times to obtain the pure recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity.

[0014] A third aspect of the present invention provides the use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein described above in the fields of neural circuit structure and function analysis and detection of neural network loss and reconstruction.

[0015] A fourth aspect of the present invention provides the use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein in the field of vaccine and drug research and development.

[0016] A fifth aspect of the present invention provides the use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein described above in the field of establishing a disease model of pseudorabies virus infection.

[0017] A sixth aspect of the present invention provides the use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein described above in the field of research on viral replication or pathogenic mechanism.

[0018] Compared with the existing technology, the technical solution provided by the present invention has at least the following advantages:

[0019] The present invention provides a recombinant pseudorabies virus that highly sensitively expresses red fluorescent protein for retrograde transsynaptic labeling, as well as a preparation method and application thereof. The recombinant pseudorabies virus solves the problem of low efficiency in expressing red fluorescence in existing methods and has broad application value and market prospects in the analysis of neural circuit structure and function, vaccine and drug development, disease model establishment, detection of neural network loss and reconstruction, and research on viral replication or pathogenic mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of a plasmid containing homology arms and highly efficient expression of red fluorescent protein;

[0021] FIG2 is a fluorescence imaging diagram of the recombinant pseudorabies virus PRV803 efficiently expressing red fluorescent protein;

[0022] FIG3 is a signal diagram of primary motor cortical neurons at the injection site of PRV803 infection;

[0023] FIG4 is a signal diagram of PRV803 retrograde transsynaptic infection of contralateral primary motor cortical neurons;

[0024] FIG5 is a diagram showing that PRV803 virus efficiently transduces primary motor cortical neurons across multiple synapses from the periphery;

[0025] Figure 6 shows that PRV803 virus efficiently transduces neurons of the paraventricular nucleus of the hypothalamus across multiple synapses from the periphery. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to specific embodiments.

[0027] Experimental procedures

[0028] 1. Construction of a plasmid containing homology arms and highly efficient expression of red fluorescent protein

[0029] In order to obtain a plasmid capable of homologous recombination with PRV, the corresponding left homologous arm (LHA) and right homologous arm (RHA) were inserted into the upstream and downstream of the pCAG-EGFP-CW3SL plasmid (donated by Brinkase (Shenzhen) Biotechnology Co., Ltd.). The LHA sequence (as shown in SEQ ID NO. 1) and the RHA sequence (as shown in SEQ ID NO. 2) were gene synthesized. The LHA sequence was double-digested with EcoRI and MluI and then ligated and inserted into the pCAG-EGFP-CW3SL plasmid that had been double-digested with the same enzymes to obtain the pLHA-CAG-EGFP-CW3SL plasmid. The RHA sequence was double-digested with SpeI and HindIII and then ligated and inserted into the pLHA-CAG-EGFP-CW3SL plasmid that had been double-digested with the same enzymes to obtain the pLHA-CAG-EGFP-CW3SL-RHA plasmid. The mScarletI-T2A-mScarletI-P2A-mScarletI sequence (as shown in SEQ ID NO.3), and after double digestion with AgeI and EcoRV, ligated and inserted into the pLHA-CAG-EGFP-CW3SL-RHA plasmid that had been double digested with the same enzymes, a plasmid containing homology arms and highly efficient expression of red fluorescent protein was obtained, named pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid. The plasmid map of pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA is shown in Figure 1. All PCR primers, gene synthesis, and sequencing in the present invention were performed by Beijing Qingke Biotechnology Co., Ltd.

[0030] 2. Establishment of a recombinant pseudorabies virus expressing highly sensitive red fluorescence

[0031] 2 μg of the newly constructed pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid was transfected into BHK-21 cells using the lipofectamine transfection method. After 5 hours, fresh complete medium (Dulbecco's modified eagle medium, DMEM) containing 2% (volume fraction) fetal bovine serum (FBS) was replaced, and 1 μl of PRV-Bartha virus (titer of 1×10 7PFU / ml) were used. Cytopathic effects and fluorescent protein expression were observed at different time periods. After 48 hours, the supernatant was collected, diluted 100-fold, and infected with BHK-21 cells. One hour later, the supernatant was aspirated and inoculated with complete medium containing agar. After 48 hours, plaque purification was performed. Red fluorescent plaques were picked and inoculated onto BHK-21 cells cultured in fresh DMEM supplemented with 2% FBS. After 48 hours, the supernatant was collected and diluted again for infection and plaque purification. After repeating this process four times, pure, highly sensitive, red fluorescent recombinant pseudorabies virus (PRV803) was obtained. The PRV803 viral supernatant was added to BHK-21 cells pre-cultured in 10 cm dishes to expand the virus. After 48 hours, the supernatant was collected, combined, filtered through a 0.45 μm sterile filter membrane, and the supernatant was collected. The supernatant was then centrifuged at 50,000 g for approximately 2.5 hours in a high-speed centrifuge (Beckman) pre-cooled to 4°C. The supernatant after centrifugation was removed in the clean bench, and 1 ml of PBS solution (pre-cooled in a 4°C refrigerator) was added to dissolve the virus. The collected virus was further concentrated and purified using a 20% sucrose solution. The concentration conditions were the same as the above-mentioned primary concentration method. Finally, the supernatant was slowly removed with a pipette, and the precipitate was resuspended with a certain amount of PBS and placed in a desktop centrifuge. It was centrifuged at 8000 g and 4°C for 15 minutes to remove some residual impurities. The supernatant was slowly collected, aliquoted, and stored in a -80°C refrigerator. The titer was determined using a plaque assay. The titer of the concentrated PRV803 virus was 2×10 9 PFU / ml.

[0032] In order to verify that the efficiency of expressing red fluorescence in the present invention is significantly better than that of the existing system, equal amounts (1 ul each) of PRV803 virus and PRV724 virus (Front Neuroanat. 2019; 13: 63.) were taken to infect BHK-21 cells (PRV803 virus as the experimental group and PRV724 virus as the control group, and the titer of both was diluted to 2×10 7 PFU / ml), then placed in a 37 ° C, 5% (v / v) CO2 incubator for further culture, and then fluorescence imaging was performed using the same exposure parameters. The results are shown in Figure 2, where the left figure in Figure 2 is red fluorescence imaging after recombinant pseudorabies virus PRV724 infected BHK-21 cells, with an exposure time of 300ms; the right figure is red fluorescence imaging after recombinant pseudorabies virus PRV803 infected BHK-21 cells, with an exposure time of 300ms. Under the same exposure conditions, the fluorescence brightness of the right figure is significantly better than that of the left figure, that is, the efficiency of PRV803 prepared by the present invention in expressing red fluorescent protein is significantly higher than that of PRV724 (Front Neuroanat. 2019; 13: 63.).

[0033] 3. In vivo application of recombinant pseudorabies virus expressing highly sensitive red fluorescence

[0034] (1) Prepared PRV803 (100 nL / mouse, titer 2×10 9 The virus (1000 PFU / ml) was injected stereotaxically into the primary motor cortex (M1) of 8-10-week-old C57BL / 6 mice (purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd.). Three days after infection, the mice were anesthetized by intraperitoneal injection of 0.5 ml of a 1% sodium pentobarbital-0.9% sodium chloride solution. The brain tissue was then dissected by cardiac perfusion using phosphate buffered saline (PBS) and 4% paraformaldehyde solution (PFA). The mouse brain tissue was fixed with PFA solution overnight and dehydrated with 30% sucrose-PBS solution. The dehydrated brain tissue was thoroughly embedded in tissue embedding medium and cut into 40 μm thick brain slices using a freezing microtome. The slices were imaged using an Olympus VS120 slide scanning microscope (Olympus, Japan). The results of in vivo detection showed that the recombinant pseudorabies virus PRV803 can infect the primary motor cortex neurons at the injection site as shown in Figure 3, and efficiently retrogradely transsynaptically infect neurons in upstream brain regions, such as the contralateral primary motor cortex as shown in Figure 4.

[0035] (2) 2.5 μl of PRV803 virus was injected into the hind limb muscles of 8-10 week old C57BL / 6 mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.). Five days later, the brains were perfused and imaged. The results of in vivo detection showed that PRV803 virus can efficiently transduce brain cells from the periphery across multiple synapses, including primary motor cortex neurons as shown in Figure 5 and hypothalamic paraventricular nucleus neurons as shown in Figure 6.

[0036] Those skilled in the art will appreciate that the above-described embodiments are specific examples for implementing the present application, and that in actual applications, various changes in form and detail may be made thereto without departing from the spirit and scope of the present application. Any person skilled in the art may make changes and modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application shall be subject to the scope defined in the claims.

[0037] The nucleotide sequence of the present invention is as follows:

Claims

1. A recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity, characterized in that: The recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity is obtained by packaging a plasmid containing homology arms and highly efficient expression of red fluorescent protein; Among them, the plasmid containing homology arms and efficiently expressing red fluorescent protein is pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid.

2. The method for preparing a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity according to claim 1, characterized in that: The method comprises the following steps: 1) Using the pCAG-EGFP-CW3SL plasmid as a vector, inserting the left homology arm into the upstream of the pCAG-EGFP-CW3SL plasmid and the right homology arm into the downstream of the pCAG-EGFP-CW3SL plasmid, to obtain the pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid; wherein the left homology arm is denoted as LHA and the right homology arm is denoted as RHA; 2) The pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid was transfected into BHK-21 cells, which were then infected with PRV-Bartha virus, and the cell culture medium supernatant was collected; after purification, a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity was obtained.

3. The method for preparing a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity according to claim 2, characterized in that: The step 1) specifically includes: Genetically synthesizing LHA sequence and RHA sequence, and inserting the LHA sequence into the upstream of the pCAG-EGFP-CW3SL plasmid that has been double-digested with EcoRI and MluI, thereby obtaining pLHA-CAG-EGFP-CW3SL plasmid; and then inserting the RHA sequence into the downstream of the pLHA-CAG-EGFP-CW3SL plasmid that has been double-digested with SpeI and HindIII, thereby obtaining pLHA-CAG-EGFP-CW3SL-RHA plasmid; The mScarletI-T2A-mScarletI-P2A-mScarletI sequence was gene synthesized, and the mScarletI-T2A-mScarletI-P2A-mScarletI sequence was double-digested with AgeI and EcoRV and then connected and inserted into the pLHA-CAG-EGFP-CW3SL-RHA plasmid that had been double-digested with the same enzymes to obtain the pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid.

4. The method for preparing a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity according to claim 3, characterized in that: The nucleotide sequence of the LHA sequence is shown in SEQ ID NO: 1; the nucleotide sequence of the RHA sequence is shown in SEQ ID NO:

2.

5. The method for preparing a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity according to claim 3, characterized in that: The nucleotide sequence of the mScarletI-T2A-mScarletI-P2A-mScarletI sequence is shown in SEQ ID NO:

3.

6. The method for preparing a recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity according to claim 2, characterized in that: The step 2) specifically includes: The pLHA-CAG-mScarletI-T2A-mScarletI-P2A-mScarletI-CW3SL-RHA plasmid is transfected into BHK-21 cells by a liposome transfection method. After 5 hours, a fresh complete medium containing 2% fetal bovine serum is replaced, and PRV-Bartha virus is added. After 48 hours, the supernatant is collected, and the BHK-21 cells are infected after dilution. After 1 hour, the supernatant is removed and a complete medium containing agar is added. After 48 hours, plaques are picked and purified. Empty plaques with red fluorescence are picked and transferred to BHK-21 cells in a fresh complete medium containing 2% fetal bovine serum. After 48 hours, the supernatant is collected and diluted for infection and plaque picking and purification are continued. After repeating 4 times, the pure recombinant pseudorabies virus expressing red fluorescent protein with high sensitivity is obtained.

7. Use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein as claimed in claim 1 in the field of neural circuit structure and function analysis, and detection of neural network loss and reconstruction.

8. Use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein as claimed in claim 1 in the field of vaccine and drug research and development.

9. Use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein as claimed in claim 1 in the field of establishing a disease model of pseudorabies virus infection.

10. Use of the highly sensitive recombinant pseudorabies virus expressing red fluorescent protein as claimed in claim 1 in the field of research on virus replication or pathogenic mechanism.

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