Cell line for efficiently proliferating EnvA pseudotype rabies virus as well as preparation method and application of cell line

Abstract

The invention provides a cell line for efficiently proliferating EnvA pseudotype rabies virus as well as a preparation method and application of the cell line. Specifically, the invention provides a BHK cell line in which an exogenous expression cassette for expressing EnvA and a fluorescent reporter gene is integrated in a cell genome, and a method for preparing the cell line. The cell line can be used for packaging the EnvA pseudotype rabies virus, and the generated EnvA pseudotype rabies virus can be used for marking and tracking specific types of neurons and is used for researching neural pathways.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a cell line for the efficient propagation of EnvA pseudorabies virus, its preparation method, and its applications. Background Technology

[0002] Rabies virus (RV) is a commonly used tracing tool by neuroscientists. Wild-type rabies virus belongs to the genus Lyssavirus in 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. Through reverse genetics, the glycoprotein gene G, responsible for viral invasion, was deleted from the genome, resulting in the first-generation rabies virus (ΔG). The RV envelope glycoprotein (GP) is essential for its retrograde transsynaptic movement. Its receptor is abundantly distributed at the axon terminals. After infection, it can retrogradely enter the neuronal cell body along the axon to initiate viral replication. RV lacking the G protein (RV-ΔG) loses its transsynaptic ability, but its replication and transcription are unaffected (it can sustain high-abundance expression of exogenous genes). The first-generation rabies virus had two applications: First, ΔG viruses coated with the original glycoprotein B19G could reverse label neurons, achieving a reverse labeling function. Second, by exogenously expressing the B19G glycoprotein and TVA (a cell surface receptor specific to birds, not endogenously expressed in mammalian nervous systems) in the initiating cells of interest, and simultaneously using rabies virus coated with EnvA (an outer membrane protein of avian sarcoma virus that specifically recognizes the TVA receptor), reverse "cross-monosynaptic" tracking of specific initiating cells could be achieved. Because mammalian cells lack the EnvA receptor TVA, recombinant deletion-type rabies virus coated with EnvA could not directly infect mammalian cells. Only when exogenous TVA is expressed on specific nerve cells can the EnvA-coated rabies virus vector specifically infect these nerve cells. After replication and transcription, the virus crosses the synapse under the coating of B19G to infect the next level of neurons. However, since the upstream neurons do not express B19G, the virus jumps only to this level of neurons and cannot jump to the next level of neurons. This is called "cross-synaptic tracking".

[0003] By using Cre transgenic mice and Cre-LoxP to control the expression of AAV helper viruses containing TVA and G proteins, it is possible to express TVA and G proteins only in specific types of neurons in specific regions, thereby achieving reverse transmonosynaptic labeling of specific types of neurons using RV-ΔG (EnvA). This method has been widely used in neural pathway research; however, the biggest challenge of this technology is the need for large-scale propagation of EnvA pseudorabies virus to obtain high-titer viruses for animal experiments. However, currently reported similar cell lines have low viral titers, therefore, there is a need in the field for cell lines capable of more efficiently propagating EnvA pseudorabies virus. Summary of the Invention

[0004] The purpose of this invention is to provide a cell line capable of efficiently proliferating EnvA pseudorabies virus, a method for preparing the cell line, and the uses of the cell line.

[0005] In a first aspect of the invention, a cell line for the efficient propagation of EnvA pseudotyped rabies virus is provided, said cell line being a BHK cell line, wherein the cell genome of said cell line integrates an exogenous expression cassette expressing EnvA and a fluorescent reporter gene.

[0006] In another preferred embodiment, the cell line used for efficient propagation of EnvA pseudorabies virus is a polyclonal cell line.

[0007] In another preferred embodiment, the nucleotide sequence of the EnvA gene is shown in SEQ ID NO:1.

[0008] In another preferred embodiment, the amino acid sequence of the EnvA protein encoded by the EnvA gene is shown in SEQ ID NO:2.

[0009] In another preferred embodiment, the fluorescent reporter gene includes EGFP, GFP, BFP, mCherry, tdTomato, or a combination thereof.

[0010] In another preferred embodiment, the fluorescent reporter gene is EGFP.

[0011] In another preferred embodiment, the nucleotide sequence of the fluorescent reporter gene EGFP is shown in SEQ ID NO:3.

[0012] In another preferred embodiment, the amino acid sequence of EGFP encoded by the fluorescent reporter gene EGFP is shown in SEQ ID NO:4.

[0013] In another preferred embodiment, the exogenous expression cassette has the structure shown in Formula I:

[0014] Z1-Z2-Z3-Z4-Z5 (I)

[0015] In the formula,

[0016] Z1 is a promoter or a 5'-UTR element containing a promoter;

[0017] Z2 is an optional enhancer;

[0018] Z3 is the nucleotide sequence of the EnvA gene;

[0019] Z4 is the nucleotide sequence of the fluorescent reporter gene; and

[0020] Z5 is a zero or 3'-UTR element.

[0021] In another preferred embodiment, Z2 is an internal ribosome entry site (IRES).

[0022] In another preferred embodiment, one or more copies of the exogenous expression cassette are integrated into the genome of the cell line.

[0023] In another preferred embodiment, the cell line expresses moderate fluorescence, wherein "moderate fluorescence" means that the fluorescence intensity is the second highest in the entire sorted cell population, and such cells account for 4-5% of the entire sorted cell population.

[0024] In another preferred embodiment, the supernatant titer of the EnvA pseudotyped rabies virus produced packaged on the cell line is greater than 1E6 iu / mL, preferably greater than 2E6 iu / mL.

[0025] In another preferred embodiment, the EnvA pseudotyped rabies virus is an EnvA-coated rabies virus lacking the G protein (RV-ΔG).

[0026] In another preferred embodiment, the EnvA pseudotyped rabies virus is used to label specific neurons expressing the VA protein, thereby enabling reverse "cross-single-level synapse" tracking of neurons.

[0027] In a second aspect of the invention, a method for preparing a cell line for efficient propagation of EnvA pseudotyped rabies virus as described in the first aspect of the invention is provided, the method comprising the following steps:

[0028] (a) Preparation of viral vectors containing exogenous expression cassettes expressing EnvA and fluorescent reporter genes;

[0029] (b) Infecting BHK cells with the viral vector obtained from step (a) to obtain infected BHK cells; and

[0030] (c) The infected BHK cells from step (b) are passaged and expanded, and then flow cytometry is used to screen for BHK cell populations expressing moderate fluorescence intensity, which is the cell line used for efficient proliferation of EnvA pseudorabies virus.

[0031] In another preferred embodiment, step (a) further includes the following steps:

[0032] (a1) Construct a target gene plasmid containing an exogenous expression cassette expressing EnvA and a fluorescent reporter gene; and

[0033] (a2) The target gene plasmid and the viral packaging plasmid are transduced into host cells, and the host cells are cultured to obtain a viral vector containing an exogenous expression cassette expressing EnvA and a fluorescent reporter gene.

[0034] In another preferred embodiment, the target gene plasmid is a plasmid EnvA-IRES-EGFP containing an exogenous expression cassette expressing the EnvA and EGFP genes.

[0035] In another preferred embodiment, the target gene plasmid is as follows: Figure 1 As shown.

[0036] In another preferred embodiment, the nucleotide sequence of the EnvA gene is shown in SEQ ID NO:1.

[0037] In another preferred embodiment, the amino acid sequence of the EnvA protein encoded by the EnvA gene is shown in SEQ ID NO:2.

[0038] In another preferred embodiment, the fluorescent reporter gene includes EGFP, GFP, BFP, mCherry, tdTomato, or a combination thereof.

[0039] In another preferred embodiment, the fluorescent reporter gene is EGFP.

[0040] In another preferred embodiment, the nucleotide sequence of the fluorescent reporter gene EGFP is shown in SEQ ID NO:3.

[0041] In another preferred embodiment, the amino acid sequence of EGFP encoded by the fluorescent reporter gene EGFP is shown in SEQ ID NO:4.

[0042] In another preferred embodiment, the viral vector is a retroviral vector.

[0043] In another preferred embodiment, the viral packaging plasmid includes: gag / pol plasmid and pCAG-VSVG plasmid.

[0044] In another preferred embodiment, the host cells in step (a2) are selected from: HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0045] In another preferred embodiment, in step (a2), the target gene plasmid and viral packaging plasmid are transduced into host cells using a cell transfection reagent.

[0046] In another preferred embodiment, in step (a2), the cell transfection reagent is Lipofectamine. TM 2000.

[0047] In another preferred embodiment, in step (a2), the ratio of plasmid DNA to cell transfection reagent during transduction is 1:1-3, more preferably 1:2-3, and even more preferably 1:2.3.

[0048] In another preferred embodiment, in step (b), the infection multiplicity (MOI) of the infection is 200 to 500, more preferably 300 to 400; and even more preferably about 300.

[0049] In another preferred embodiment, in step (b), BHK cells are inoculated in a 96-well plate with the viral vector obtained from step (a), wherein the number of BHK cells is 15-25K cells per well, preferably about 20K cells per well.

[0050] In another preferred embodiment, in step (c), the infected BHK cells are passaged and expanded 24-48 hours after infection;

[0051] Specifically, the passage expansion refers to: 24-48 hours after infection, the infected BHK cells in the 96-well plate are expanded into the 24-well plate; once the cells have grown into a confluent monolayer in the 24-well plate (i.e., the cell confluence reaches more than 90%), the cells are expanded into 10cm culture dishes for further culture.

[0052] In another preferred embodiment, in step (c), the infected BHK cells grow into a monolayer in a 10cm culture dish (i.e., the cell confluence reaches more than 90%), and screening begins.

[0053] In another preferred embodiment, in step (c), flow cytometry sorting includes: sorting the fluorescently positive BHK cells detected by the flow cytometry into two cell collection groups based on fluorescence intensity, namely a high-intensity fluorescence group and a medium-intensity fluorescence group. The high-intensity fluorescence group refers to the cells with the highest fluorescence intensity, accounting for 1%-2% (preferably 1%-1.5%, more preferably 1.2%-1.5%) of the entire sorted cell population, and the medium-intensity fluorescence group refers to the cells with the second highest fluorescence intensity, accounting for 4%-6% (preferably 4%-5%, more preferably 4.5%-5%) of the entire sorted cell population. The cell population of the medium-intensity fluorescence group is the target cell line.

[0054] In a third aspect of the invention, a method for packaging EnvA pseudotyped rabies virus is provided, the method comprising packaging using a cell line as described in the first aspect of the invention.

[0055] In another preferred embodiment, the method includes the steps of:

[0056] (S1) Infect the cell line as described in the first aspect of the invention with G protein-deficient rabies virus (RV-ΔG); and

[0057] (S2) Culture the infected cell line to obtain EnvA pseudorabies virus.

[0058] In another preferred embodiment, the EnvA pseudotyped rabies virus is an EnvA-coated rabies virus lacking the G protein (RV-ΔG).

[0059] In another preferred embodiment, the G protein-deficient rabies virus (RV-ΔG) is a recombinant RV-ΔG coated with the original glycoprotein B19G, namely RV-ΔG(BG19).

[0060] In another preferred embodiment, the EnvA pseudotyped rabies virus is obtained by replacing the B19G shell in RV-ΔG (BG19) with the EnvA shell in a cell line as described in the first aspect of the invention.

[0061] In another preferred embodiment, step (S1) specifically includes the following steps:

[0062] (S1a) One day before infection, the cell line as described in the first aspect of the present invention is seeded into cell culture dishes at a seeding density of 1E6 to 3E6 / 10cm culture dish, preferably 1E6 to 2E6 / 10cm culture dish; more preferably, 1.5E6 / 10cm culture dish; and

[0063] (S1b) On the day of infection, the cells in the culture dish were counted, and RV-ΔG virus was inoculated into the culture dish to infect the cells at an appropriate multiplicity of infection.

[0064] In another preferred embodiment, step (S2) specifically includes the following steps:

[0065] (S2a) Culture the infected cell line, wash the cells 2-3 times with buffer at 24 hours and 48 hours post-infection, and replenish with fresh cell culture medium; and

[0066] (S2b) On days 3 to 5 post-infection, cell culture supernatant was collected to obtain EnvA pseudorabies virus.

[0067] In another preferred embodiment, the method further includes step (S3): determining the titer of the obtained EnvA pseudotyped rabies virus.

[0068] In another preferred embodiment, the viral titer is determined using the FACS method.

[0069] In another preferred embodiment, the supernatant titer of the obtained EnvA pseudotyped rabies virus is greater than 1E6 iu / mL, preferably greater than 2E6 iu / mL.

[0070] In another preferred embodiment, the EnvA pseudotyped rabies virus is used to label specific neurons expressing the VA protein, thereby enabling reverse "cross-synaptic" tracking of neurons.

[0071] In a fourth aspect of the invention, the use of the cell line as described in the first aspect of the invention is provided for packaging EnvA pseudotyped rabies virus, thereby for preparing a reagent for reverse “cross-monosynaptic” tracing of neurons.

[0072] In a fifth aspect of the invention, an EnvA pseudotyped rabies virus is provided, which is prepared by the method described in the third aspect of the invention.

[0073] In another preferred embodiment, the EnvA pseudotyped rabies virus is used as a reagent for preparing reverse "cross-monosynaptic" tracing of neurons.

[0074] 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

[0075] Figure 1 The target gene plasmid snapgene map is displayed.

[0076] Figure 2The image shows the expression of EnvA in BHK cells. The top image is an image of BHK-EnvA before sorting under a fluorescence microscope. The bottom image shows the cell line with stable fluorescence expression obtained by flow cytometry sorting, named BHK-EnvA. Scale bar: 200 μm. Left: Bright field. Right: EGFP.

[0077] Figure 3 The growth curves of RVΔG-4mCherry(EnvA) packaged on BHK-EnvA cells are shown.

[0078] Figure 4 This image shows mCherry expression in RVΔG-4mCherry (EnvA) under a fluorescence microscope. The images are taken on days 3, 4, and 5 after inoculation at MOI=2. Scale bar: 200 μm. Left: EGFP, expressed by the BHK-EnvA cell line; Right: mCherry, expressed by the RVΔG-4mCherry virus.

[0079] Figure 5 The growth curves of RVΔG-4mCherry (EnvA) from day 3 to day 5 in the packaging experiment are shown. Data from 10 replicate experiments were collected, with cell numbers ranging from 4.8E6 to 1.2E7 / 15cm culture dish at inoculation. Viral supernatants were harvested 3-5 days after inoculation, and the titer of each supernatant was calculated. Detailed Implementation

[0080] Through extensive and in-depth research, the inventors have, for the first time, constructed a cell line for the efficient propagation of EnvA pseudotyped rabies virus. This cell line is a polyclonal cell line, with its genome integrated with exogenous expression cassettes expressing EnvA and a fluorescent reporter gene. It can be used to encapsulate G protein-deficient rabies virus that produces the EnvA coat. The supernatant titer of the EnvA pseudotyped rabies virus packaged in this cell line is consistently above 1E6 IU / mL, thus enabling efficient propagation of the EnvA pseudotyped rabies virus for reverse trans-monosynaptic labeling and tracking of specific types of neurons expressing the TVA protein.

[0081] Based on this, the present invention was completed.

[0082] the term

[0083] 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.

[0084] 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.).

[0085] 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”.

[0086] EnvA pseudorabies virus

[0087] As used herein, the terms "EnvA pseudotyped rabies virus," "Rabies virus lacking the G protein in the EnvA shell," "Rabies virus lacking the G protein coated with EnvA," and "RV-ΔG(EnvA)" are used interchangeably and all refer to recombinant rabies viruses coated with an EnvA shell and lacking the G protein in their viral genome. In one specific embodiment of the present invention, the above terms refer to the recombinant rabies virus RVΔG-4mCherry(EnvA) prepared in the examples, which is produced using the BHK-EnvA cell line of the present invention, by replacing the B19G shell of the RVΔG-4mCherry(B19G) recombinant rabies virus with an EnvA shell. The EnvA pseudotyped rabies virus produced by the present invention can be used to label neurons expressing TVA, enabling reverse cross-synaptic tracing of neurons.

[0088] As used in this paper, the terms "G protein-deficient rabies virus," "ΔG virus," and "RV-ΔG" are used interchangeably, all referring to recombinant rabies viruses whose genome has deleted the glycoprotein gene G responsible for viral invasion. Furthermore, the ΔG virus coated with the original glycoprotein B19G is designated RV-ΔG(BG19), which can retrogradely label neurons, achieving a retrograde labeling function. As used in this paper, the term "RVΔG-4mCherry(B19G)" refers to a recombinant rabies virus in which the glycoprotein G gene in the RV genome has been replaced with the mCherry red fluorescent protein gene, but the original glycoprotein B19G is used for viral packaging, and the viral coat remains the B19G glycoprotein.

[0089] Cell lines for efficient propagation of EnvA pseudorabies virus

[0090] In one aspect of the invention, a cell line for the efficient propagation of EnvA pseudotyped rabies virus is provided, said cell line being a BHK cell line whose genome integrates an exogenous expression cassette expressing EnvA and a fluorescent reporter gene (e.g., EGFP). In a specific embodiment of the invention, said cell line is named the BHK-EnvA cell line, whose genome integrates an exogenous expression cassette expressing EnvA and EGFP.

[0091] The BHK-EnvA cell line of this invention is a polyclonal cell line, a group of cells expressing moderate fluorescence obtained through flow cytometry sorting. Compared to monoclonal cells, which require multiple generations of cell replication to form a cell population, polyclonal cell lines integrate exogenous genes more stably, and the overall cell condition (viability, survival rate, etc.) is also relatively better. Therefore, the BHK-EnvA cell line of this invention can be used for efficient propagation of EnvA pseudorabies virus, and the supernatant titer of the packaged EnvA pseudorabies virus is consistently above 1E6 IU / mL.

[0092] As used in this invention, the terms "cell line of the present invention", "cell line for efficient propagation of EnvA pseudorabies virus", "cell line as described in this invention", "cell line for efficient propagation of EnvA pseudorabies virus of the present invention", and "cell line for efficient propagation of EnvA pseudorabies virus as described in this invention" are used interchangeably and all refer to the BHK cell line as described in the first aspect of this invention.

[0093] Methods for constructing cell lines for efficient propagation of EnvA pseudorabies virus

[0094] In another aspect of the invention, a method for preparing a cell line for efficient propagation of EnvA pseudotyped rabies virus as described herein is also provided, the method comprising the following steps:

[0095] (a) Preparation of viral vectors containing exogenous expression cassettes expressing EnvA and fluorescent reporter genes;

[0096] Specifically, it includes the following steps:

[0097] (a1) Construct a target gene plasmid containing an exogenous expression cassette expressing EnvA and a fluorescent reporter gene; preferably, the target gene plasmid is a plasmid EnvA-IRES-EGFP containing an exogenous expression cassette expressing EnvA and EGFP genes; and

[0098] (a2) The target gene plasmid and the viral packaging plasmid are transduced into host cells, and the host cells are cultured to obtain a viral vector containing an exogenous expression cassette expressing EnvA and a fluorescent reporter gene;

[0099] Preferably, the target gene plasmid and viral packaging plasmid are transduced into host cells using a cell transfection reagent, wherein the ratio of plasmid DNA to cell transfection reagent during transduction is 1:1-3, more preferably 1:2-3, and even more preferably 1:2.3.

[0100] (b) Infect BHK cells with the viral vector obtained from step (a) to obtain infected BHK cells;

[0101] Specifically, based on an infection multiplicity (MOI) of 200–500, preferably 300–400; more preferably about 300, BHK cells are seeded in 96-well plates, with a cell number of 15–25K cells per well, preferably about 20K cells per well; and

[0102] (c) The infected BHK cells in step (b) are passaged and expanded, and then flow cytometry is used to screen and obtain a population of BHK cells expressing moderate fluorescence intensity, which is the cell line used for efficient proliferation of EnvA pseudorabies virus.

[0103] Specifically, 24-48 hours after infection, the infected BHK cells were passaged and expanded, that is, the infected BHK cells in the 96-well plates were expanded into 24-well plates; when the cells reached a confluence of more than 90% in the 24-well plates, the cells were expanded into 10 cm culture dishes for further culture; when the infected BHK cells reached a confluence of more than 90% in the 10 cm culture dishes, the selection process began;

[0104] The flow cytometry sorting process involves separating fluorescently positive BHK cells detected by the instrument into two cell collection groups based on fluorescence intensity: a high-intensity fluorescence group and a medium-intensity fluorescence group. The high-intensity fluorescence group consists of cells with the highest fluorescence intensity, accounting for 1%-2% (ideally 1%-1.5%, more preferably 1.2%-1.5%) of the entire sorted cell population. The medium-intensity fluorescence group consists of cells with the second highest fluorescence intensity, accounting for 4%-6% (ideally 4%-5%, more preferably 4.5%-5%) of the entire sorted cell population. The cell population in the medium-intensity fluorescence group is the target cell line.

[0105] Packaging EnvA pseudorabies virus

[0106] This invention also provides a method for packaging EnvA pseudotyped rabies virus, using a cell line as described in this invention. Specifically, the method includes the following steps:

[0107] (S1) Infect the cell line as described in this invention with G protein-deficient rabies virus (RV-ΔG); and

[0108] (S2) Culture the infected cell line to obtain EnvA pseudorabies virus.

[0109] In one specific embodiment of the present invention, the method includes: infecting the BHK-EnvA cell line of the present invention with RVΔG-4mCherry (B19G); culturing the infected cell line, washing the cells with buffer 2-3 times at 24 hours and 48 hours after infection, and replenishing with fresh cell culture medium; and collecting the cell culture supernatant from day 3 to day 5 after infection to obtain EnvA pseudotyped rabies virus RVΔG-4mCherry (EnvA).

[0110] application

[0111] The present invention also provides the use of the cell line as described herein for packaging EnvA pseudorabies virus, the resulting EnvA pseudorabies virus being used to prepare reagents for reverse "cross-monosynaptic" tracing of neurons.

[0112] The sequence information involved in this invention:

[0113] EnvA nucleotide sequence (SEQ ID NO:1)

[0114]

[0115] EnvA amino acid sequence (SEQ ID NO: 2)

[0116] MEAVIKAFLTGHPGKVSKKDSKKKPPATSKKDPEKTPLLPTRVNYIIGVLVLCEVTGVRADVHLLEQPGNLWITWANRTGQTDFCLSTQSATSPFQTCLIGIPSPISEGDFKGYVSDNCTTLEPHRLVSRGIPGGPENSTTLTYQKVSCLLLKLNVSLLDEPSELQLLGSQSLPNITNITRIPSVAGGCIGFTPYDSPAGVYGWDRREVTHILLTDPGNNPFFDKASNSSKPFTVVTADRHNLFMGSEYCGAYGYRFWEMYNCSQMRQNWSICQDVWGRGPPENWCTSTGGTWVNQSKEFNETAPFSFTVNCTGSNLGNVSGCCGEPITILPPEAWVDSTQGSFTKPKALPPAIFLICGDRAWQGIPSRPVGGPCYLGKLTMLAPKHTDILKVLVNSSRTGIRRKRSTSHLDDTCSDEVQLWGPTARIFASILAPGVAAAQALREIERLACWSVKQANLTTSLLGDLLDDVTSIRHAVLQNRAAIDFLLLAHGHGCEDVAGMCCFNLSDHSESIQKKFQLMKEHVNKIGVDSDPIGSWLRGLFGGIGEWAVHLLKGLLLGLVVILLLVVCLPCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGETRL*

[0117] EGFP nucleotide sequence (SEQ ID NO: 3)

[0118] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0119] Amino acid sequence of EGFP (SEQ ID NO: 4)

[0120] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0121] The main advantages of this invention include:

[0122] (1) The cell line of the present invention is a polyclonal cell line. Compared with monoclonal cells, which require multiple generations of cell replication to form a cell population, polyclonal cell lines integrate exogenous genes more stably.

[0123] (2) The cell line of the present invention can efficiently proliferate EnvA pseudorabies virus. Compared with the prior art, the supernatant titer of EnvA pseudorabies virus packaged in the cell line of the present invention is stable at above 1E6 iu / mL.

[0124] (3) The cell lines of the present invention have good reproducibility. The preparation method described in the present invention can stably obtain cell lines with good performance and can be used to efficiently proliferate EnvA pseudorabies virus.

[0125] (4) The EnvA pseudotyped rabies virus produced by the cell line packaging of the present invention can be used to label specific types of neurons expressing TVA protein, thereby achieving reverse cross-level synaptic tracking of neurons.

[0126] 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.

[0127] Example 1: Preparation of BHK-EnvA cell line

[0128] 1.1 Virus Packaging Plasmid

[0129] Plasmid EnvA-IRES-GFP: Based on a retroviral vector (Moloney murine leukemia virus, GenBank: AF033811.1), the target sequence of the EnvA gene was inserted (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2). After obtaining the target sequence by PCR, the vector was digested with NotI, and the two were ligated using the HiFi homologous recombination method (see...). Figure 1 The sequence was confirmed using Sanger sequencing; plasmid gag / pol: contains genes for proteins required for viral packaging, including the viral core protein and enzymes required for viral replication; plasmid pCAG-VSVG: encodes envelope proteins.

[0130] 1.2 Cells

[0131] Both HEK-293T cells and BHK cells were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0132] 1.3 Experimental Procedure

[0133] (1) Preparation of EnvA-expressing retroviruses:

[0134] a) Two days before transfection, coat the culture dish with PLL (Poly-L-lysine solution, SIGMA-P4832, diluted 1:5 before use). That is, add 5ml to a new 15cm culture dish, gently shake to spread it evenly in the culture dish, and incubate overnight at 37°C.

[0135] b) One day before transfection, approximately 1.8E7 HEK-293T cells were seeded in culture dishes coated with PLL the day before.

[0136] c) On the day of transfection, confirm that the cells have reached 70-80% confluency, and use DNA:L2K (Lipofectamine) TM 293T cells were transfected using 2000 transfection reagent (Invitrogen, catalog number: 11668019) at a ratio of 1:2.3, with endotoxin-free plasmids (in one 15cm culture dish: target plasmid EnvA-IRES-GFP 22.5ug, plasmid gag / pol 16.9ug, pCAG-VSVG 5.6ug). During DNA and L2K incubation, the culture medium in the 15cm culture dish was replaced with 12ml. Medium (serum-reduced medium, Gibco, catalog number: 11058021), after 5-6 hours of transfection, replace each culture dish with 12 ml of DMEM complete medium containing 10% fetal bovine serum (Dulbecco's Modified Eagle Medium, Gibco, catalog number: C11995500BT).

[0137] d) Harvest the supernatant three days after transfection. After centrifugation at 2000 rpm for 5 minutes, take the supernatant and filter it with a 0.45-μm Stericup filter (BIOFIL, catalog number: FCF000007). Then, concentrate it by high-speed centrifugation (22000 rpm, 4℃ for 2 hours) to obtain concentrated virus.

[0138] (2) Preparation of cell lines: The concentrated virus was seeded into a 96-well plate with a BHK cell count of about 20K (200ul of medium per well) at MOI=300. After 24 hours, the fluorescence was observed and the medium was changed. After 1-2 days, the cells in the 96 wells were expanded into 24-well plates, and the cells were further passaged and expanded.

[0139] (3) Cell line sorting: BHK-EnvA cell line was screened by flow cytometry.

[0140] The specific method of flow sorting is as follows:

[0141] a) Cell digestion: Once the cells have grown into a confluent monolayer in a 10cm culture dish, wash and discard the cells with 3ml of DPBS (phosphate-buffered saline, Gibco, catalog number: 14190144), then digest the cells with 2ml of trypsin (Gibco, catalog number: 25300054) at room temperature for 2min; then add 4ml of DMEM containing 10% fetal bovine serum to terminate the digestion.

[0142] b) Centrifugation: After resuspending the cells, centrifuge at 200g for 5 minutes at room temperature;

[0143] c) DNA removal: After centrifugation, discard the supernatant, add 360ul DMEM to resuspend the cells, and add 40ul DNase I Solution (SIGMA, catalog number: D4513, storage concentration 1mg / mL, final concentration used 100ug / mL) to remove extracellular DNA. Incubate at room temperature for 15min.

[0144] d) Centrifugation: After incubation, add 5 ml of DMEM to resuspend the cells and centrifuge at 200 g for 5 min at room temperature;

[0145] e) Sorting: After centrifugation, resuspend the cells in 1 ml of DMEM and pass them through a 35 μm flow cytometer. 12x75mm test tubes (with cell sieve caps, catalog number: 352235) were used to filter and prepare single-cell suspensions for direct flow cytometry sorting. Two cell collection populations were plotted based on fluorescence intensity: a high-intensity fluorescence group (top 1.2%) and a medium-intensity fluorescence group (4.5%). The resulting medium-intensity positive cells were named BHK-EnvA. After the cells reached confluent monolayers, they were passaged for expansion and preservation. The percentages mentioned above refer to the percentage of the entire sorted cell population.

[0146] 1.4 Results and Conclusions

[0147] Green fluorescence expression was observed in cells infected with the virus, indicating that the EnvA gene had been successfully integrated into BHK cells (see...). Figure 2 However, the fluorescence intensity varies among different cells.

[0148] In cells with high fluorescence intensity, high fluorescence indicates a higher number of integrated gene copies. Because the integration is random, it can affect the basic functions of the cell and may not be beneficial to viral replication.

[0149] Low fluorescence in cells with low intensity fluorescence indicates low gene copy number and low protein expression, which leads to low viral titer.

[0150] If single-clone cells are obtained through sorting, the cells need to replicate for many generations to grow into a population of cells, which can lead to instability of the integrated genes.

[0151] Therefore, the cell lines were sorted using flow cytometry, and cells with stable EGFP and EnvA expression and moderate fluorescence were obtained. These cells were named BHK-EnvA and were used for subsequent virus preparation tests.

[0152] Example 2: Validation of the packaging efficiency of EnvA pseudotyped rabies virus in the BHK-EnvA cell line

[0153] 2.1 Experimental Materials

[0154] Cell line: BHK-EnvA cell line obtained in Example 1.

[0155] Virus: RVΔG-4mCherry(B19G), viral titer 3.40E9 iu / mL.

[0156] 2.2 Experimental Procedure

[0157] (1) RV virus proliferation in the EnvA outer shell:

[0158] a) One day before inoculation, BHK-EnvA cells of good growth at 1.5E6 were passaged into two 10cm culture dishes.

[0159] b) On the day of inoculation, take out a culture dish and count the cells in it. Inoculate RVΔG-4mCherry(B19G) (virus titer 3.40E9 iu / mL) according to MOI=2.

[0160] c) 24 hours after inoculation, wash the cells twice with 5 ml of DPBS, and then add 7 ml of DMEM containing 10% fetal bovine serum to continue culturing.

[0161] d) 48 hours after inoculation, wash the cells twice with 5 ml of DPBS, and then add 7 ml of DMEM containing 10% fetal bovine serum to continue culturing.

[0162] e) From day 3 to day 10, collect the supernatant daily. 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 at -20°C.

[0163] (2) Determination of RV virus titer in the EnvA capsid after proliferation: The supernatant was inoculated into 293T-TVA950-BFP cells (a cell line expressing the EnvA-specific receptor TVA950), and the viral titer was determined using the FACS method. The specific steps of this method are as follows:

[0164] a) One day before inoculation, seed 293T-TVA950-BFP cells in 96-well plates at 30K / well.

[0165] b) On the day of receiving the virus, dilute the supernatant according to Table 1 below:

[0166] Table 1

[0167] Dilution Adding samples diluent 0 33ul Viral Supernatant 297ul 10% DMEM 1 33ul virus supernatant "0" 297ul 10% DMEM 2 33ul virus supernatant "1" 297ul 10% DMEM

[0168] c) Remove the original cell culture medium from the 96-well plate and inoculate each diluted sample into the 96-well plate with 200 μL of the cell culture medium.

[0169] d) 72 h after inoculation, aspirate the supernatant from the 96-well plate and wash each well with 100 μL of DPBS. After removing 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.

[0170] 2.3 Results

[0171] In this experiment, supernatant harvesting began on day 3 and was conducted once a day until day 10 after inoculation, resulting in the collection of a total of 8 supernatants (sup1-sup8).

[0172] The viral titer of the daily harvested supernatant was determined (see Table 2), and a viral growth curve was plotted (see Table 2). Figure 3 Based on the viral growth curve, the BHK-EnvA cell line's ability to package the virus is mainly concentrated on days 3-5, i.e., sup1-sup3. Therefore, when performing EnvA recoating experiments on the virus, it is only necessary to collect the supernatant from days 3-5 (sup1-sup3). Regarding the fluorescence expression after inoculation, good mCherry expression of RVΔG-4mCherry can be observed from days 3-5 after inoculation (see...). Figure 4 ).

[0173] Table 2 Viral titers in supernatant of BHK-EnvA cell lines

[0174]

[0175] Subsequently, using the invented cell line, the virus was inoculated at MOI=2, and the RVΔG-4mCherry (EnvA) was packaged 10 times repeatedly. The results showed stable repeatability, and by adjusting the number of cells at inoculation, different titers of supernatant were produced (see Table 3). Virus growth curves were also plotted (see Table 3). Figure 5 The average titer of sup1 supernatant was 2.7E6 iu / mL (from 7.8E5 to 1.1E7 iu / mL), the average titer of sup2 supernatant was 2.8E6 iu / mL (from 8.5E5 to 6.0E6 iu / mL), and the average titer of sup3 supernatant was 1.7E6 iu / mL (from 5.0E5 to 3.9E6 iu / mL).

[0176] Table 3. Virus titers in supernatant of BHK-EnvA cell line in 10 RVΔG-4mCherry(EnvA) packaging experiments

[0177]

[0178] 2.4 Conclusion

[0179] The BHK-EnvA cell line of this invention can efficiently proliferate EnvA pseudotyped rabies virus, effectively replacing the outer shell of RVΔG-4mCherry (B19G) virus from B19G to EnvA; and the titers of the first three harvested supernatants can be consistently above the 6th power, with the average titer of sup1 supernatant being 2.7E6 iu / mL, the average titer of sup2 supernatant being 2.8E6 iu / mL, and the average titer of sup3 supernatant being 1.7E6 iu / mL.

[0180] 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. A cell line for the efficient propagation of EnvA pseudotyped rabies virus, characterized in that, The cell line is the BHK cell line, and the cell genome of the cell line integrates an exogenous expression cassette expressing EnvA and a fluorescent reporter gene.

2. The cell line as described in claim 1, characterized in that, The cell line used for efficient propagation of EnvA pseudotyped rabies virus is a polyclonal cell line.

3. The cell line as described in claim 1, characterized in that, The fluorescent reporter genes include EGFP, GFP, BFP, mCherry, tdTomato, or combinations thereof.

4. The cell line as described in claim 1, characterized in that, The exogenous expression cassette has the structure shown in Formula I: Z1-Z2-Z3-Z4-Z5(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 of the EnvA gene; Z4 is the nucleotide sequence of the fluorescent reporter gene; and Z5 is a zero or 3'-UTR element.

5. The cell line as described in claim 1, characterized in that, The cell line expresses moderate fluorescence, where "moderate fluorescence" means that the fluorescence intensity is the second highest in the entire sorted cell population, and such cells account for 4-5% of the entire sorted cell population.

6. A method for preparing a cell line for efficient propagation of EnvA pseudotyped rabies virus as described in any one of claims 1-5, characterized in that, The method includes the following steps: (a) Preparation of viral vectors containing exogenous expression cassettes expressing EnvA and fluorescent reporter genes; (b) Infecting BHK cells with the viral vector obtained from step (a) to obtain infected BHK cells; and (c) The infected BHK cells from step (b) are passaged and expanded, and then flow cytometry is used to screen for BHK cell populations expressing moderate fluorescence intensity, which is the cell line used for efficient proliferation of EnvA pseudorabies virus.

7. The method as described in claim 6, characterized in that, In step (c), flow cytometry sorting includes: sorting the fluorescently positive BHK cells detected by the flow cytometry into two cell collection groups based on fluorescence intensity, namely a high-intensity fluorescence group and a medium-intensity fluorescence group. The high-intensity fluorescence group refers to the cells with the highest fluorescence intensity, accounting for 1%-2% (preferably 1%-1.5%, more preferably 1.2%-1.5%) of the entire sorted cell population, and the medium-intensity fluorescence group refers to the cells with the second highest fluorescence intensity, accounting for 4%-6% (preferably 4%-5%, more preferably 4.5%-5%) of the entire sorted cell population. The cell population in the medium-intensity fluorescence group is the target cell line.

8. A method for packaging EnvA pseudotyped rabies virus, characterized in that, The method includes packaging using a cell line as described in any one of claims 1-5.

9. The method as described in claim 8, characterized in that, The method includes the following steps: (S1) Infect the cell line as described in any one of claims 1-5 with G protein-deficient rabies virus (RV-ΔG); and (S2) Culture the infected cell line to obtain EnvA pseudorabies virus.

10. Use of the cell line according to any one of claims 1-5 for packaging EnvA pseudotyped rabies virus, thereby for preparing a reagent for reverse "across monosynaptic" tracing of neurons.

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