High-yield rabies virus cell line as well as preparation method and application thereof

Abstract

The invention provides a high-yield rabies virus cell line as well as a preparation method and application thereof. Specifically, the invention provides an engineered cell for producing the recombinant rabies virus, the recombinant rabies virus with effective titer can be produced by using the cell virus through one-time passage, and a neural circuit tracing tool with excellent performance is provided for the application of the defective rabies virus in neural network reverse labeling. The research in the brain science field is promoted.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a cell line that produces high levels of rabies virus, its preparation method, and its applications. Background Technology

[0002] The brain's neural network is a complex structure composed of a vast number of neurons with diverse morphologies and characteristics, connected by synapses. This neural network is the foundation for the brain's specific functions. Currently, the mechanisms of normal physiological activities and brain diseases are unclear due to a lack of information about the connections within the brain's neural networks. Revealing the connections between functional neurons is a fundamental problem in neuroscience research and is crucial for understanding the pathological mechanisms of normal brain function and nervous system diseases. In neuroscience, especially when using mice as research subjects, scientists commonly use neural tracking viruses to study neural circuits. Therefore, high-performance neural circuit tracking tools are urgently needed in brain science research.

[0003] Rabies virus is a commonly used tracing tool by neuroscientists. It is widely used in the study of neural pathways, but the biggest challenge at present is that the viral titer is not high during passage, so the virus needs to be passaged many times to reach the ideal titer. However, rabies virus will produce many mutations during multiple passages, causing some of the virus to lose its replication function, which will reduce the labeling efficiency in animal experiments.

[0004] Therefore, there is an urgent need in this field to develop a cell line for the efficient propagation of rabies virus, as well as its preparation method and applications. Summary of the Invention

[0005] The purpose of this invention is to provide a cell line for the efficient propagation of rabies virus, its preparation method, and its application.

[0006] In a first aspect, the present invention provides an engineered cell for producing recombinant rabies virus, wherein the engineered cell is a BHK cell, and the genome of the BHK cell is integrated with an exogenous B19G expression cassette for expressing the B19G protein.

[0007] In another preferred embodiment, the B19G protein is a glycoprotein of the SAD-B19 rabies virus.

[0008] In another preferred embodiment, the nucleotide sequence of said B19G is shown in SEQ ID No:1.

[0009] In another preferred embodiment, the amino acid sequence of the B19G protein is shown in SEQ ID No:2.

[0010] In another preferred embodiment, the engineered cells are selected from the group consisting of BHK-21 cells, BHK cells, and BSR-T7 / 5 cells.

[0011] In another preferred embodiment, the recombinant rabies virus is a reverse-labeled virus.

[0012] In another preferred embodiment, the recombinant rabies virus is used to label neurons in a neural circuit.

[0013] In another preferred embodiment, the engineered cells can produce recombinant rabies virus with a supernatant titer of 6E6-2E8 iu / mL after one passage of the virus; more preferably 1E7-2E8 iu / mL; and even more preferably 2E7-2E8 iu / mL.

[0014] In another preferred embodiment, one or more copies of the B19G expression cassette are integrated into the genome of the engineered cell.

[0015] In another preferred embodiment, the genome of the engineered cell further integrates additional expression cassettes selected from the group consisting of: gag / pol expression cassettes, marker protein expression cassettes, pCAG-VSVG, or combinations thereof.

[0016] In another preferred embodiment, the exogenous B19G expression cassette has the structure of Formula I:

[0017] Z1-Z2-Z3-Z4(I)

[0018] In the formula,

[0019] Z1 is a promoter or a 5'-UTR element containing a promoter;

[0020] Z2 is an optional enhancer;

[0021] Z3 is the nucleotide sequence encoding the B19G protein (SEQ ID NO:1); and

[0022] Z4 is a zero or 3'-UTR element.

[0023] In another preferred embodiment, the B19G may or may not have a tag sequence.

[0024] In another preferred embodiment, the promoter is a CMV promoter.

[0025] In another preferred embodiment, the enhancer is a CMV, i.e., an early enhancer.

[0026] In another preferred embodiment, the marker protein is a green fluorescent protein.

[0027] In another preferred embodiment, the engineered cells can promote the proliferation of recombinant rabies virus.

[0028] In another preferred embodiment, under the same or substantially the same culture conditions, the ratio (T1 / T0) of the rabies virus titer of the engineered cells to that of the wild cells is ≥20, preferably ≥50, and more preferably ≥100.

[0029] In another preferred embodiment, the wild-type cells are selected from cells containing the B19G expression cassette from the group consisting of HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa cells; preferably the HEK293-T cell line.

[0030] In another preferred embodiment, the engineered cells can promote the proliferation of recombinant rabies virus, with a supernatant titer of 6E6-2E8 iu / mL; more preferably 1E7-2E8 iu / mL; and even more preferably 2E7-2E8 iu / mL.

[0031] In a second aspect of the present invention, a method for constructing engineered cells that produce high levels of recombinant rabies virus is provided, the method comprising the following steps:

[0032] (a) Preparation of a viral vector containing the nucleotide sequence of the B19G gene;

[0033] (b) Infect BHK cells with the viral vector obtained from (a) to obtain engineered cells that produce high levels of recombinant rabies virus.

[0034] In another preferred embodiment, step (a) includes the following steps:

[0035] (a1) Transfect a plasmid containing the B19G gene and a viral packaging plasmid into a host cell, and culture the host cell to obtain a viral vector containing the nucleotide sequence of the B19G gene.

[0036] In another preferred embodiment, the viral vector is a retroviral vector.

[0037] In another preferred embodiment, the nucleotide sequence of the B19G gene is shown in SEQ ID NO: 1.

[0038] In another preferred embodiment, the plasmid containing the B19G gene is a plasmid B19G-IRES-EGFP containing the coding sequence for the green fluorescent protein EGFP.

[0039] In another preferred embodiment, the viral packaging plasmid is one or more selected from the group consisting of:

[0040] (1) A plasmid containing nucleic acid encoded by the relevant proteins gag / pol required for virus preparation;

[0041] (2) Plasmids containing nucleic acids encoded by the envelope protein CAG-VSVG.

[0042] In another preferred embodiment, in step (a1), the host cell is selected from the group consisting of HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa cell lines.

[0043] In another preferred embodiment, step (a1) further includes: transfecting HEK293-T cells with the plasmid and transfection reagent at a ratio of DNA:transfection reagent = 1:1 to 3 (preferably 1:2 to 3; more preferably 1:2.3), collecting the supernatant and filtering it after 48h to 120h to obtain the viral vector.

[0044] In another preferred embodiment, the transfection reagent is Lipofectamine. TM 2000 transfection reagent.

[0045] In another preferred embodiment, in step (b), the BHK cells are BHK-21 cells.

[0046] In another preferred embodiment, in step (b), the infection multiplicity (MOI) of the infection is 300 to 700, more preferably 400 to 600, and even more preferably about 500.

[0047] In another preferred embodiment, step (b) further includes: infecting BHK-21 cells with the viral vector at a multiplicity of infection (MOI) of 300–700, observing cell state and fluorescence ratio after 24–72 h, sorting positive cells by flow cytometry and passaged to obtain engineered cells BHK-B19G that produce high levels of recombinant rabies virus.

[0048] In another preferred embodiment, the term "passage" refers to 1-20 generations, more preferably 1-10 generations, or even one generation.

[0049] In a third aspect of the invention, a method for producing recombinant rabies virus is provided, wherein the engineered cells described in the first aspect of the invention are cultured to obtain recombinant rabies virus.

[0050] In another preferred embodiment, the recombinant rabies virus is used to label neurons.

[0051] In a fourth aspect of the invention, a recombinant rabies virus for reverse labeling is provided, said recombinant rabies virus being produced from the engineered cells described in the first aspect of the invention.

[0052] In another preferred embodiment, the recombinant rabies virus is capable of retrogradely infecting nerve cells from the axon terminals of neurons to achieve retrograde labeling of neurons.

[0053] In a third aspect of the invention, the use of engineered cells as described in the first aspect of the invention is provided for the preparation of neural marker formulations.

[0054] In another preferred embodiment, a neuronal reverse labeling agent is used to prepare a neural circuit.

[0055] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0056] Figure 1 This is the target gene plasmid sequence map in this invention. The vector cloning method includes PCR purification and recovery of the target sequence, digestion of the vector with NotI enzyme, and ligation of the two using the HiFi seamless ligation method; the sequence is confirmed by Sanger sequencing.

[0057] Figure 2 This shows the expression of B19G retrovirus in BHK cells. Before FACS, the images are taken under a fluorescence microscope after BHK cells were infected with the retrovirus at MOI=500 and before BHK-B19G cells were sorted. After FACS, the images show the cell lines that stably expressed the retrovirus, sorted by flow cytometry and named BHK-B19G. Scale bar: 200 μm. The left side shows the bright field results. Green indicates EGFP expression results.

[0058] Figure 3 This image shows the mCherry expression results of RVΔG-4mCherry (B19G) in the BHK-B19G or 293T-B19G cell lines under a fluorescence microscope. From top to bottom, the images are taken on days 2, 5, and 8 post-infection. Scale bar: 200 μm. Green represents EGFP results, expressed in either the BHK-B19G or 293T-B19G cell lines. Red represents mCherry expression by the RVΔG-4mCherry virus.

[0059] Figure 4This is a growth curve of RVΔG-4mCherry (B19G). The rabies virus expressing mCherry was propagated in BHK-B19G or 293T-B19G cells. Viral supernatants were harvested 1-10 days post-infection, denoted as Sup1-Sup10, and the titer of each supernatant was calculated. The blue line represents the viral titer produced by RVΔG-4mCherry packaged in BHK-B19G cells over 10 consecutive days. The red line represents the viral titer produced by RVΔG-4mCherry packaged in 293T-B19G cells over 10 consecutive days. Unit: iu / mL.

[0060] Figure 5 This is a growth curve of RVΔG-4mCherry detected in 5 independent experiments. The blue line represents the viral supernatant harvested 1-6 days after inoculation in 5 independent experiments (MOI=0.2), labeled Sup1-Sup6, and the titer of each supernatant was calculated. Unit: iu / mL.

[0061] Figure 6 This is a diagram of the stereotactic injection pattern in the brain and the labeling of RVΔG-4mCherry virus from the VPM region of the retrograde cortical neurons; anterior-posterior axis (AP), medial-lateral axis (ML), dorsal-venteal axis (DV), scale bar 200μm. Detailed Implementation

[0062] Through extensive and in-depth research, including numerous screenings and tests, the inventors have provided engineered cells that promote the proliferation of recombinant rabies virus and their preparation method. Unexpectedly, the inventors discovered that the engineered cells BHK-B19G provided by this invention produce sufficiently high viral titers with only one passage, making them ideal for the production of first-generation rabies virus. Furthermore, the viral titer of BHK-B19G cells is more than 100 times that of 293T-B19G cells, approximately 1E7-2E8 IU / mL, significantly increasing the viral titer. Moreover, the cell culture conditions are easy to achieve and control, and the cost is low; the produced recombinant rabies virus remains stable even after multiple passages. Based on these findings, this invention was completed.

[0063] the term

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0065] As used in this article, the terms "neural circuit" and "neural loop" are used interchangeably.

[0066] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0067] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0068] As used herein, the terms “room temperature” or “normal temperature” refer to a temperature of 4–40°C, preferably 25 ± 5°C.

[0069] Rabies virus (RV)

[0070] Rabies virus (RV) belongs to the genus Lyssavirus of the family Rhabdoviridae. It has an envelope and its genome is a single-stranded, negative-sense RNA. The viral genome is approximately 12 kb long, with five genes—N, P, M, G, and L—arranged sequentially from the 3′ to 5′ ends. These genes encode nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and transcriptase protein, respectively. In neuroscience research, the vector used is based on the rabies virus vaccine strain SAD B19. Through reverse genetics, the glycoprotein gene G, responsible for viral invasion, was deleted from the genome, resulting in the first-generation rabies virus (RV-ΔG). The ΔG virus, coated with the original glycoprotein B19G, can retrolabel neurons, achieving a reverse labeling function.

[0071] Wild-type rabies virus (RV) is the causative agent of zoonotic rabies, possessing transmissible properties in the nervous system and exhibiting high pathogenicity in both humans and animals. Existing recombinant rabies viruses are derived from the rabies virus vaccine strain (SADB-19). With the advancement of reverse genetics techniques, modified RVs can be used for neural circuit research. After infecting the central nervous system, RVs primarily label neurons, with almost no labeling of glial cells; infected neurons show almost no significant lesions or lysis within a certain timeframe (7-12 days). Currently, RV systems can be divided into reverse labeling systems and reverse transsynaptic systems.

[0072] Recombinant rabies virus

[0073] As used herein, the terms "first-generation rabies virus," "recombinant rabies virus," "RV-ΔG," and "reverse-labeled virus" are used interchangeably and all refer to the recombinant rabies virus produced using the engineered cells described in the first aspect of this invention.

[0074] The glycoprotein (G) of the RV is essential for its retrograde transsynaptic function. RVs lacking G protein (RV-ΔG) lose their transsynaptic function, but their replication and transcription are unaffected (they can continuously express exogenous genes at high abundance). Therefore, when RV-ΔG carries a reporter gene (such as the EGFP gene of this invention), it can retrogradely mark the fine morphology of neurons with high brightness.

[0075] B19G gene expression cassette

[0076] The engineered cells of the present invention comprise an exogenous B19G expression cassette, which has the structure of Formula I:

[0077] Z1-Z2-Z3-Z4(I)

[0078] In the formula,

[0079] Z1 is a promoter or a 5'-UTR element containing a promoter;

[0080] Z2 is an optional enhancer;

[0081] Z3 is the nucleotide sequence encoding the B19G protein; and

[0082] Z4 is a zero or 3'-UTR element.

[0083] Optionally, the B19G may or may not have a tag sequence.

[0084] This invention, through extensive and innovative experiments, has discovered that BHK engineered cells can promote the proliferation of recombinant rabies virus. Surprisingly, the inventors found that the viral load of BHK-B19G cells is more than 100 times that of 293T-B19G cells; the viral load is 1E7-2E8 IU / mL, significantly increasing the viral load of cells used for producing recombinant rabies virus. Furthermore, the cell culture conditions are easy to achieve and control, and the cost is low; the produced recombinant rabies virus can be mass-produced after a single passage. The engineered cells provided by this method have broad practical application value and promising prospects.

[0085] Engineered cells for producing recombinant rabies virus

[0086] An engineered cell for producing recombinant rabies virus is a BHK-B19G cell, wherein the engineered cell is a BHK cell, and the genome of the BHK cell integrates an exogenous B19G expression cassette for expressing the B19G protein. Preferably, the B19G protein is selected from the glycoprotein of SAD-B19 rabies virus.

[0087] Preferably, the nucleotide sequence of the B19G is shown in SEQ ID No:1. The amino acid sequence of the B19G protein is shown in SEQ ID No:2.

[0088] Preferably, the engineered cells are selected from BHK-21 cells.

[0089] Preferably, the BHK-B19G cells promote the proliferation of recombinant rabies virus.

[0090] Preferably, the recombinant rabies virus is used to label neurons.

[0091] Preferably, the engineered cells can produce an effective titer of recombinant rabies virus after only one passage.

[0092] Preferably, the effective titer refers to the viral titer that can effectively label neurons.

[0093] Preferably, one or more copies of the B19G expression cassette are integrated into the genome of the engineered cell.

[0094] Preferably, the genome of the engineered cell further integrates additional expression cassettes selected from the group consisting of: gag / pol expression cassette, EGFP expression cassette, pCAG-VSVG, or combinations thereof.

[0095] Unless otherwise specified, the specific sequence of the expression cassette of the present invention is known to those skilled in the art.

[0096] A method for constructing engineered cells that produce high-yield recombinant rabies virus.

[0097] The method for constructing engineered cells that produce high-yield recombinant rabies virus in this invention includes the following steps:

[0098] (a) Preparation of a viral vector containing the nucleotide sequence of the B19G gene;

[0099] (b) Infect BHK cells with the viral vector obtained from (a) to obtain engineered cells that produce high levels of recombinant rabies virus.

[0100] Optionally, step (a) includes the following steps:

[0101] (a1) Transfect a plasmid containing the B19G gene and a viral packaging plasmid into a host cell, and culture the host cell to obtain a viral vector containing the nucleotide sequence of the B19G gene.

[0102] Optionally, the viral vector is a retroviral vector.

[0103] Optionally, the nucleotide sequence of the B19G gene is shown in SEQ ID NO: 2.

[0104] Optionally, the plasmid containing the B19G gene is a plasmid containing the green fluorescent protein EGFP, namely B19G-IRES-EGFP.

[0105] Optionally, the viral packaging plasmid is selected from one or more of the following groups:

[0106] (1) A plasmid containing nucleic acid encoded by the relevant proteins gag / pol required for virus preparation;

[0107] (2) Plasmids containing nucleic acids encoded by the envelope protein CAG-VSVG.

[0108] Optionally, in step (a1), the host cell is selected from the group consisting of HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa cell lines.

[0109] Optionally, step (a1) further includes: transfecting 293T cells with the plasmid and transfection reagent at a ratio of DNA:transfection reagent = 1:1 to 3 (preferably 1:2 to 3; more preferably 1:2.3), collecting the supernatant and filtering it after 48h to 120h to obtain the viral vector.

[0110] Optionally, the transfection reagent is Lipofectamine. TM 2000 transfection reagent.

[0111] Optionally, in step (b), the BHK cells are selected from BHK-21 cells.

[0112] Optionally, in step (b), the infection multiplicity (MOI) of the infection is 300 to 700, more preferably 400 to 600, and even more preferably about 500.

[0113] Optionally, step (b) further includes: infecting BHK-21 cells with the viral vector at a multiplicity of infection (MOI) of 300–700, observing cell status and fluorescence ratio after 24–72 hours, sorting positive cells by flow cytometry and passaged to obtain engineered cells BHK-B19G that produce high levels of recombinant rabies virus.

[0114] Optionally, the term "generation" refers to 1-20 generations, more preferably 1-10 generations, or even one generation.

[0115] The main advantages of this invention include:

[0116] (1) The present invention constructs the cell line BHK-B19G, which only requires one passage of the virus to obtain a first-generation rabies virus with a sufficiently high titer, greatly reducing the production cost of the virus; it also reduces the possibility of mutation of the rabies virus during multiple passages and improves the labeling efficiency of reverse labeling neurons.

[0117] (2) The BHK-B19G cell line constructed in this invention has a stable process for producing rabies virus, and the batch-to-batch differences in virus products are small.

[0118] (3) The cell culture conditions provided by the present invention are easy to implement and control, and have low cost.

[0119] (4) The toxin production titer of BHK-B19G cells provided by the present invention is more than 100 times that of 293T-B19G cells, approximately 1E7-2E8 iu / mL, which greatly improves the toxin production titer of cells.

[0120] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0121] Example 1: Preparation of the BHK-B19G cell line

[0122] 1. Construction of plasmid B19G-IRES-EGFP:

[0123] Based on a retroviral vector (Moloney murine leukemia virus, GenBank: AF033811.1), the target sequence was the B19G gene (see SEQ ID NO: 1). After PCR of the target sequence, the vector was digested with NotI enzyme; the two were ligated using a HiFi seamless ligation method (see...). Figure 1 The B19G-IRES-EGFP plasmid was obtained; plasmid gag / pol: related protein genes required for virus preparation; pCAG-VSVG: encoding envelope protein.

[0124] 2. Preparation of B19G retrovirus:

[0125] a) Two days before transfection, add 5 ml of proportionally diluted PLL (Poly-L-lysine solution, SIGMA, catalog number: P4832, storage concentration 0.01%, before use, dilute with 40 ml of DPBS to 8 ml of PLL solution at a ratio of 1:5) to two new 15 cm culture dishes, shake well and spread evenly in the dishes, and incubate overnight at 37°C.

[0126] b) One day before transfection, remove the 15cm culture dish that has been incubated with PLL overnight and discard the PLL solution; then passage HEK 293T cells into it at a number of approximately 1.5-1.8E7 / dish, ensuring that the cells reach 70-80% confluency at the time of transfection the next day.

[0127] c) On the day of transfection, confirm that the cell density reaches 70-80% confluency, and use DNA:L2K (Lipofectamine) TM The Invitrogen 2000 transfection reagent (catalog number: 11668019) was used at a ratio of 1:2.3. Endotoxin-free plasmids were used to transfect 293T cells (Chinese Academy of Sciences Cell Bank). During the DNA-transfection reagent incubation process, the culture medium in the 15cm culture dish was replaced with 12ml. Medium (serum-reduced medium, Gibco, catalog number: 11058021) should be replaced with 12 ml of DMEM complete medium (Dulbecco's Modified Eagle Medium, Gibco, catalog number: C11995500BT) containing 10% fetal bovine serum in each culture dish after 5-6 hours.

[0128] d) Harvest the supernatant three days after transfection. After centrifugation at 2000 rpm for 5 minutes, take the supernatant and filter it through a 0.45-μm Stericup filter (BIOFIL, catalog number: FCF000007). Then, concentrate it by high-speed centrifugation (22000 rpm, 4 degrees for 2 hours) to obtain concentrated B19G retrovirus.

[0129] 3. Preparation of cell lines:

[0130] The concentrated B19G retrovirus was seeded into 96-well plates containing approximately 10K BHK-21 cells (Chinese Academy of Sciences Cell Bank) at an MOI of 500. Fluorescence was observed after 24 hours, and the culture medium was replaced with fresh medium as needed. After 1-2 days, the cells in the 96-well plates were expanded into 24-well plates, and the cells were subsequently passaged and expanded.

[0131] 4. Cell line sorting:

[0132] The BHK-B19G cell line was screened using flow cytometry. The specific flow cytometry method is as follows:

[0133] Once the cells have reached a confluence of 10cm in the culture dish, discard the cell culture medium. Wash once with 3ml of DPBS (phosphate-buffered saline, Gibco, catalog number: 14190144), discard the wash, and then digest the cells with 2ml of trypsin (Gibco, catalog number: 25300054) at room temperature for 2 minutes, or gently shake the culture dish to observe cell movement. Add 4ml of DMEM containing 10% fetal bovine serum to stop the digestion. Resuspend the cells and transfer them to a 15ml centrifuge tube. Centrifuge at 200g for 5 minutes at room temperature. After centrifugation, discard the supernatant, add 360ul of DMEM to resuspend the cells, and add 40ul of DNase I Solution (SIGMA, catalog number: D4513, stock concentration 1mg / mL, final concentration 100ug / mL) to remove extracellular DNA. Incubate at room temperature for 15 minutes. After incubation, add another 5ml of DMEM to resuspend the cells and centrifuge at 200g for 5 minutes at room temperature. After centrifugation, rinse with 1ml of DMEM. Cells were resuspended in DMEM and passed through flow cytometry tubes with a pore size of 35 μm. The 12x75mm test tubes (with cell sieve caps, catalog number: 352235) were used to filter the single-cell suspension, which was then directly subjected to flow cytometry sorting. Positive cells were obtained after sorting and named BHK-B19G. After the cells reached a confluent monolayer, they were passaged, expanded, and cryopreserved.

[0134] Experimental results are as follows Figure 2 As shown, green fluorescence expression was observed in the virus-infected cells, indicating that the virus was successfully prepared and could successfully infect BHK cells. Due to the high multiplicity of infection of the virus used, the proportion of positive cells was relatively high, but a small number of negative cells were still present. Subsequently, the cell lines were sorted using flow cytometry, and gradient sorting was performed based on the expression intensity of EGFP to obtain positive cells with high EGFP expression levels, which were used for subsequent virus preparation and testing.

[0135] Example 2: Detection of the ability of BHK-B19G cell line to proliferate rabies virus

[0136] 2.1 Experimental Materials:

[0137] (1) Cell lines: BHK-B19G cells, 293T-B19G cells;

[0138] (2) Virus: RVΔG-4mCherry(B19G), viral titer was 8.68E7i.u. / mL.

[0139] 2.2 RV virus replication:

[0140] 1) Two days before inoculation, add 3 ml of 1:5 diluted PLL (dilute 0.01% PLL solution with 5 times the volume of DPBS) to two new 10 cm culture dishes and incubate overnight at 37°C.

[0141] 2) The day before receiving the drug,

[0142] a. Remove the 10cm culture dish that has been incubated with PLL overnight, completely aspirate the PLL solution, and passage the same number of 293T-B19G cells in good growth condition into the two culture dishes respectively, ensuring that the cells reach 80% confluence when inoculated the next day.

[0143] b. Take two more 10cm culture dishes that have not been incubated with PLL, and passage the same number of BHK-B19G cells in good growth condition into each of the two culture dishes, ensuring that the cells reach 80% confluence when inoculated the next day.

[0144] 3) On the day of inoculation, after confirming that the cell density to be inoculated reaches 80%, take out one culture dish containing 293T-B19G cells or BHK-B19G cells, count each type of cell in the culture dish, and inoculate RVΔG-4mCherry(B19G) (virus titer 8.68E7 iu / mL) into the other two uncounted cell dishes according to MOI=0.1.

[0145] 4) Collect the supernatant daily for 1-10 days after inoculation. Add 7 ml of DMEM containing 10% fetal bovine serum to the culture dish. Filter the harvested supernatant through a 0.45-μm Stericup filter and store it at -20℃.

[0146] 2.3 Determination of RV virus titer after proliferation:

[0147] The supernatant was seeded into 293T cells, and the viral titer was determined using the FACS method. The specific FACS method is as follows:

[0148] 1) One day before inoculation, seed 293T cells at 30K / well in a 96-well plate.

[0149] 2) On the day of receiving the virus, dilute the supernatant:

[0150] "0": 33ul of virus supernatant + 297ul of DMEM, vortex to mix;

[0151] "1": 33ul of viral supernatant "0" + 297ul of DMEM, vortex to mix;

[0152] "2": 33ul of viral supernatant "1" + 297ul of DMEM, vortex to mix.

[0153] 3) Remove the original cell culture medium from the 96-well plate and inoculate each diluted sample (200 μL) into the cells of the 96-well plate.

[0154] 4) 72 h after inoculation, aspirate the supernatant from the 96-well plate and wash each well with 100 μL of DPBS. After aspirating the DPBS, add 50 μL of trypsin to each well for 2 min at room temperature, then add 100 μL of DMEM to terminate the digestion. Finally, add 50 μL of 4% PFA (Paraformaldehyde, Leagene, catalog number: DF0135 / 500 ml) to each well, wrap the 96-well plate with aluminum foil, and perform flow cytometry analysis. After obtaining the positive rate of virus-infected cells, calculate the virus titer according to the MOI formula.

[0155] 2.4 Results

[0156] Ten supernatants were collected from each group on day 10 after inoculation. The daily supernatants were used to determine the viral titer (MOI = 0.1) of RVΔG-4mCherry(B19G) expressing virus in different cell lines, and virus growth curves were plotted. The titer results are shown in Table 1.

[0157] Table 1

[0158] cell lines BHK-B19G 293T-B19G Sup1-D1 1.17E+07 1.12E+05 Sup2-D2 1.43E+08 1.14E+06 Sup3-D3 2.10E+08 3.04E+06 Sup4-D4 2.06E+08 1.85E+06 Sup5-D5 1.65E+08 9.69E+05 Sup6-D6 1.46E+08 9.40E+05 Sup7-D7 8.51E+07 8.24E+05 Sup8-D8 5.22E+07 6.23E+05 Sup9-D9 3.71E+07 7.95E+05 Sup10-D10 1.91E+07 2.53E+05

[0159] Based on the fluorescence expression after infection, on days 2, 5, and 8 post-infection, the expression of RVΔG-4mCherry mCherry in the BHK-B19G cell line was slightly better than that in the 293T-B19G cell line (see [link to data]). Figure 3 Based on the viral growth curves, the BHK-B19G cell line (supernatant titer range of 1E7-2E8 iu / mL) showed significantly better viral proliferation ability than the 293T-B19G cell line (supernatant titer range of 1E5-2E6 iu / mL) (see...). Figure 4 ).

[0160] Subsequently, the BHK-B19G cell line prepared by this invention was inoculated with virus at an MOI of 0.2, and RVΔG-4mCherry was produced 5 times. The titers of the supernatants harvested each day were measured (unit: iu / mL), and virus growth curves were plotted. The titer results are shown in Table 2.

[0161] Table 2

[0162] Number of experiments 1 2 3 4 5 Sup1-D1 5.95E+06 8.36E+06 3.67E+06 3.15E+06 3.96E+06 Sup2-D2 8.64E+07 1.07E+08 5.44E+07 3.71E+07 8.44E+07 Sup3-D3 6.96E+07 1.10E+08 9.23E+07 5.30E+07 5.62E+07 Sup4-D4 4.94E+07 1.08E+08 1.11E+08 6.35E+07 1.16E+08 Sup5-D5 3.33E+07 3.63E+07 8.64E+07 6.49E+07 9.53E+07 Sup6-D6 2.23E+07 2.48E+07 6.17E+07 4.77E+07 4.41E+07

[0163] Virus growth curve as shown Figure 5As shown, the results were reproducible and stable, and the supernatant titer remained stable at 2E7-2E8 iu / mL from the second day onwards.

[0164] The above results indicate that the viral supernatant titer after proliferation (MOI = 0.1) using the 293T-B19G cell line was 1E5-2E6 iu / mL, while the viral supernatant titer after proliferation (MOI = 0.1) using the BHK-B19G cell line of this invention was stable at 1E7-2E8 iu / mL (see [link to original text]). Figure 4 The experiment was stably repeated 5 times, and the titer of the newly proliferated (MOI=0.2) viral supernatant remained stable at 2E7-2E8 iu / mL from the second day onwards (see...). Figure 5 ).

[0165] Therefore, it can be seen that the BHK-B19G cell line of the present invention has a significantly better ability to proliferate viruses than the 293T-B19G cell line, and the ability to proliferate viruses is stable, indicating that the BHK-B19G cell line can significantly improve the proliferation efficiency of RV virus.

[0166] Example 3: Animal testing of rabies virus prepared from BHK-B19G cell line.

[0167] 1. Experimental animals: C57BL / 6 mice, 8 weeks old.

[0168] 2. A virus: RVΔG-4mCherry(B19G), viral titer 4.43E10 iu / mL.

[0169] 3. Virus testing

[0170] 1) Preparation before the experiment: Before the experiment, the virus was taken out of the -80℃ freezer and placed on ice to thaw; the mice were anesthetized with isoflurane and fixed on the stereotactic brain device, and the nose clip was attached and the mice were continuously anesthetized with isoflurane.

[0171] 2) Virus injection: such as Figure 6 As shown,

[0172] a. Make a vertical incision in the scalp using surgical scissors, and keep the incision open with forceps. Absorb any blood with a cotton ball. Removing the periosteum will allow observation of the anterior fontanelle.

[0173] b. Place the needle vertically against the anterior fontanelle, using the anterior fontanelle as the zero point of the three-dimensional coordinate system. At this point, zero out the anterior-posterior axis (AP Y-axis), medial-lateral axis (ML X-axis), and dorsoventral axis (DV Z-axis). Next, lift the needle and adjust it to (AP = -1.82, ML = -1.54). After drilling into the skull at the target point, gently puncture the meninges.

[0174] c. Use a glass electrode to draw 1 μL of virus, insert the glass electrode into the brain through the open bone hole, and reduce DV to zero as soon as it touches the dura mater, then move down to DV = -3.15.

[0175] d. The injection rate is 20 nl / min, the total injection volume is 200 nl, and the injection is stopped for 5 minutes after injection.

[0176] f. After the injection, suture the scalp and wait for the mouse to wake up before returning it to its cage.

[0177] g. Seven days after viral expression, the mice were perfused with the brain, which was then sectioned and photographed to observe the viral expression.

[0178] Experimental results are as follows Figure 6 As shown, RVΔG-4mCherry prepared using this BHK-B19G cell line can infect neurons in mice and retrogradely label neurons, and the number of labeled neurons is large.

[0179] The sequence information involved in this invention is as follows:

[0180] B19G nucleotide sequence in the BHK-B19G cell line (SEQ ID NO:1):

[0181]

[0182] The amino acid sequence of B19G protein in the BHK-B19G cell line (SEQ ID NO:2):

[0183] *

[0184] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An engineered cell for producing recombinant rabies virus, characterized in that, The engineered cells are BHK cells, and the genome of the BHK cells integrates an exogenous B19G expression cassette for expressing the B19G protein.

2. The engineered cell as described in claim 1, characterized in that, The nucleotide sequence of B19G is shown in SEQ ID No:

1.

3. The engineered cell as described in claim 1, characterized in that, The engineered cells can produce recombinant rabies virus with a supernatant titer of 6E6-2E8 iu / mL after one passage of the virus; preferably 1E7-2E8 iu / mL; more preferably 2E7-2E8 iu / mL.

4. The engineered cell as described in claim 1, characterized in that, The exogenous B19G expression cassette has the structure of Formula I: Z1-Z2-Z3-Z4(I) In the formula, Z1 is a promoter or a 5'-UTR element containing a promoter; Z2 is an optional enhancer; Z3 is the nucleotide sequence encoding the B19G protein (SEQ ID NO:1); and Z4 is a zero or 3'-UTR element.

5. The engineered cell as described in claim 1, characterized in that, The engineered cells can promote the proliferation of recombinant rabies virus, with a supernatant titer of 6E6-2E8 iu / mL; preferably 1E7-2E8 iu / mL; more preferably 2E7-2E8 iu / mL.

6. A method for constructing engineered cells that produce high-yield recombinant rabies virus, characterized in that, The method includes the following steps: (a) Preparation of a viral vector containing the nucleotide sequence of the B19G gene; (b) Infect BHK cells with the viral vector obtained from (a) to obtain engineered cells that produce high levels of recombinant rabies virus.

7. The construction method as described in claim 6, characterized in that, Step (b) further includes: infecting BHK-21 cells with the viral vector at a multiplicity of infection (MOI) of 300–700, observing cell status and fluorescence ratio after 24–72 hours, sorting positive cells by flow cytometry and passaged to obtain engineered cells BHK-B19G that produce high levels of recombinant rabies virus.

8. A method for producing recombinant rabies virus, characterized in that, The engineered cells described in claim 1 are cultured to obtain recombinant rabies virus.

9. A recombinant rabies virus for reverse labeling, characterized in that, The recombinant rabies virus is produced from the engineered cells described in claim 1.

10. The use of the engineered cells as described in claim 1, characterized in that, Used to prepare neural markers.

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