Cell lines for efficient propagation of EnvA pseudotype CVS N2c rabies virus, their preparation methods and applications

Abstract

This invention provides a cell line for the efficient propagation of EnvA pseudotyped CVS N2c rabies virus, its preparation method, and its applications. Specifically, this invention provides an N2a cell line with an exogenous expression cassette integrating EnvA and a fluorescent reporter gene into its genome, as well as a method for preparing said cell line. The cell line of this invention can be used to package EnvA pseudotyped CVS N2c rabies virus. The high-titer EnvA pseudotyped rabies virus produced can label and track specific types of neurons for neural pathway research, tracing more information about brain circuits.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a cell line for the efficient propagation of EnvA pseudotype CVS N2c rabies virus, its preparation method, and its applications. Background Technology

[0002] Neural tracing tools have been widely used for analyzing neural pathways and mapping brain atlases. Among them, rabies virus-based tools can be used for inverse transsynaptic tracing. Wild-type rabies virus 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 the 5' end. These genes encode nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and large transcriptase protein, respectively.

[0003] Typically, rabies virus tools replace the glycoprotein gene in the viral genome with a fluorescent protein gene. During viral packaging, the glycoprotein is trans-presented for complete viral packaging. To achieve nerve cell-specific infection, the transsynaptic tracing system utilizes the outer membrane protein (EnvA) receptor (TVA) of avian sarcoma virus (ASLV), specifically expressing TVA in neurons, while using the EnvA glycoprotein as the viral coat.

[0004] Scientists, in modifying the tool, compared the CVS N2c virus strain with the SAD B19 virus strain and discovered that the CVS N2c virus strain has the advantage of low virulence, allowing neurons to maintain a healthy state for a longer period. However, the long preparation time and low titer in virus preparation have hindered the widespread application of this virus strain. Therefore, there is an urgent need in the field for a cell line that can rapidly generate a high-titer CVS N2c virus strain for reverse transsynaptic tracing. Summary of the Invention

[0005] The purpose of this invention is to provide a cell line capable of efficiently proliferating EnvA pseudotype CVS N2c rabies virus, a method for preparing the cell line, and the uses of the cell line.

[0006] In a first aspect of the invention, a cell line for the efficient propagation of EnvA pseudotype CVS N2c rabies virus is provided, said cell line being an N2a cell line, and said cell line having an exogenous expression cassette expressing EnvA and a fluorescent reporter gene integrated into its cellular genome.

[0007] In another preferred embodiment, the cell line used for efficient propagation of EnvA pseudotype CVS N2c rabies virus is a polyclonal cell line.

[0008] In another preferred embodiment, the nucleotide sequence of the EnvA gene is shown in SEQ ID NO:1.

[0009] In another preferred embodiment, the amino acid sequence of the EnvA protein encoded by the EnvA gene is shown in SEQ ID NO:2.

[0010] In another preferred embodiment, the fluorescent reporter gene includes EGFP, GFP, BFP, mCherry, tdTomato, or a combination thereof.

[0011] In another preferred embodiment, the fluorescent reporter gene is EGFP.

[0012] In another preferred embodiment, the nucleotide sequence of the fluorescent reporter gene EGFP is shown in SEQ ID NO:3.

[0013] In another preferred embodiment, the amino acid sequence of EGFP encoded by the fluorescent reporter gene EGFP is shown in SEQ ID NO:4.

[0014] In another preferred embodiment, the exogenous expression cassette has the structure shown in Formula I:

[0015] Z1-Z2-Z3-Z4-Z5(I)

[0016] In the formula,

[0017] Z1 is a promoter or a 5'-UTR element containing a promoter;

[0018] Z2 is an optional enhancer;

[0019] Z3 is the nucleotide sequence of the EnvA gene;

[0020] Z4 is the nucleotide sequence of the fluorescent reporter gene; and

[0021] Z5 is a zero or 3'-UTR element.

[0022] In another preferred embodiment, Z2 is an internal ribosome entry site (IRES).

[0023] In another preferred embodiment, one or more copies of the exogenous expression cassette are integrated into the genome of the cell line.

[0024] In another preferred embodiment, the cell line expresses high-intensity fluorescence, wherein "high-intensity fluorescence" means that the fluorescence intensity accounts for the top 1.5%-2% of the total fluorescence intensity of the sorted cell population, more preferably, the top 1.2%-1.5%, more preferably, the top 1.1%-1.2%, and most preferably, the top 1.05%.

[0025] In another preferred embodiment, the supernatant titer of the EnvA pseudotype CVS N2c rabies virus packaged on the cell line is greater than 1E6 iu / mL, more preferably greater than 5E6 iu / mL, and even more preferably greater than 1E7 iu / mL.

[0026] In another preferred embodiment, the EnvA pseudotype CVS N2c rabies virus is an EnvA-coated CVSN2c rabies virus lacking the G protein (CVS N2cΔG).

[0027] In another preferred embodiment, the EnvA pseudotype CVS N2c rabies virus is used to label specific neurons expressing the TVA protein, thereby enabling reverse "cross-synaptic" tracking of neurons.

[0028] In a second aspect of the invention, a method for preparing a cell line for efficient propagation of EnvA pseudotype CVS N2c rabies virus as described in the first aspect of the invention is provided, the method comprising the following steps:

[0029] (a) Preparation of viral vectors containing exogenous expression cassettes expressing EnvA and fluorescent reporter genes;

[0030] (b) Infecting N2a cells with the viral vector obtained from step (a) to obtain infected N2a cells; and

[0031] (c) The infected N2a cells from step (b) are passaged and expanded, and then flow cytometry is used to screen and obtain a population of N2a cells expressing high fluorescence intensity, which is the cell line used for efficient proliferation of EnvA pseudotype CVS N2c rabies virus.

[0032] In another preferred embodiment, step (a) further includes the following steps:

[0033] (a1) Construct a target gene plasmid containing an exogenous expression cassette expressing EnvA and a fluorescent reporter gene; and

[0034] (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.

[0035] 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.

[0036] In another preferred embodiment, the target gene plasmid is as follows: Figure 1 As shown.

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

[0038] In another preferred embodiment, the amino acid sequence of the EnvA protein encoded by the EnvA gene is shown in SEQ ID NO:2.

[0039] In another preferred embodiment, the fluorescent reporter gene includes EGFP, GFP, BFP, mCherry, tdTomato, or a combination thereof.

[0040] In another preferred embodiment, the fluorescent reporter gene is EGFP.

[0041] In another preferred embodiment, the nucleotide sequence of the fluorescent reporter gene EGFP is shown in SEQ ID NO:3.

[0042] In another preferred embodiment, the amino acid sequence of EGFP encoded by the fluorescent reporter gene EGFP is shown in SEQ ID NO:4.

[0043] In another preferred embodiment, the viral vector is a retroviral vector.

[0044] In another preferred embodiment, the viral packaging plasmid includes: gag / pol plasmid and pCAG-VSVG plasmid.

[0045] 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.

[0046] 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.

[0047] In another preferred embodiment, in step (a2), the cell transfection reagent is Lipofectamine. TM 2000.

[0048] 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.

[0049] In another preferred embodiment, in step (b), the infection multiplicity (MOI) of the infection is 800 to 1500, more preferably 1000 to 1200; and even more preferably about 1000.

[0050] In another preferred embodiment, in step (b), N2a cells are inoculated in a 96-well plate with the viral vector obtained from step (a), wherein the number of N2a cells is 4-8K cells per well, preferably 5-6K cells per well, and more preferably 5.5K cells per well.

[0051] In another preferred embodiment, in step (c), the infected N2a cells are passaged and expanded 24-48 hours after infection;

[0052] Specifically, the passage expansion refers to: 24-48 hours after infection, the infected N2a 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.

[0053] In another preferred embodiment, in step (c), the infected N2a cells grow into a monolayer in a 10cm culture dish (i.e., the cell confluence reaches more than 90%), and screening begins.

[0054] In another preferred embodiment, in step (c), flow cytometry sorting includes: sorting the fluorescently positive N2a 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 the top 1.5%-2% of the entire sorted cell population (preferably, the top 1.2%-1.5%, more preferably, the top 1.1%-1.2%, and most preferably, the top 1.05%). The medium-intensity fluorescence group refers to the cells with the second highest fluorescence intensity, accounting for the top 3%-5% of the entire sorted cell population. The cell population of the high-intensity fluorescence group is finally sorted and is the target cell line (named N2a-EnvA-1).

[0055] In a third aspect of the invention, a method for packaging EnvA pseudotype CVS N2c rabies virus is provided, the method comprising packaging using a cell line as described in the first aspect of the invention.

[0056] In another preferred embodiment, the method includes the steps of:

[0057] (S1) Infect the cell line as described in the first aspect of the invention with G protein-deficient CVS N2c rabies virus (CVS N2cΔG); and

[0058] (S2) Culture the infected cell line to obtain EnvA pseudotype CVS N2c rabies virus.

[0059] In another preferred embodiment, the EnvA pseudotype CVS N2c rabies virus is an EnvA-coated CVSN2c rabies virus lacking the G protein (CVS N2cΔG).

[0060] In another preferred embodiment, the G protein-deficient CVS N2c rabies virus (CVS N2cΔG) is a recombinant CVS N2cΔG coated with the original glycoprotein B19G, namely CVS N2cΔG(BG19).

[0061] In another preferred embodiment, the EnvA pseudotype of CVS N2c rabies virus is obtained by replacing the B19G shell of CVS N2cΔG (BG19) with the EnvA shell in a cell line as described in the first aspect of the invention.

[0062] In another preferred embodiment, step (S1) specifically includes the following steps:

[0063] (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 5E5 to 1E6 / well, preferably 5E5 to 8E5 / well; more preferably 6E5 / well, where the aforementioned well refers to one well in a 6-well plate; and

[0064] (S1b) On the day of infection, the cells in the culture dish were counted, and CVS N2cΔG virus was inoculated into the culture dish to infect the cells at an appropriate multiplicity of infection.

[0065] In another preferred embodiment, step (S2) specifically includes the following steps:

[0066] (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

[0067] (S2b) On days 3 to 5 post-infection, cell culture supernatant was collected to obtain EnvA pseudotype CVS N2c rabies virus.

[0068] In another preferred embodiment, the method further includes step (S3): determining the titer of the obtained EnvA pseudotype CVS N2c rabies virus.

[0069] In another preferred embodiment, the method further includes step (S4): concentrating the EnvA pseudotype CVS N2c rabies virus in the harvested cell culture supernatant to obtain concentrated EnvA pseudotype CVS N2c rabies virus.

[0070] In another preferred embodiment, the viral titer is determined using the FACS method or the qPCR method.

[0071] In another preferred embodiment, the supernatant titer of the obtained EnvA pseudotyped rabies virus is greater than 1E6 iu / mL, more preferably greater than 5E6 iu / mL, and even more preferably greater than 1E7 iu / mL.

[0072] In another preferred embodiment, the concentrated EnvA pseudotype CVS N2c rabies virus titer is greater than 1E10 iu / mL, more preferably greater than 3E10 iu / mL, and even more preferably greater than 5E10 iu / mL.

[0073] In another preferred embodiment, the EnvA pseudotype CVS N2c rabies virus is used to label specific neurons expressing the TVA protein, thereby enabling reverse "cross-synaptic" tracking of neurons.

[0074] 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 pseudotype CVS N2c rabies virus, thereby for preparing a reagent for reverse “cross-monosynaptic” tracing of neurons.

[0075] In a fifth aspect of the invention, an EnvA pseudotype CVS N2c rabies virus is provided, which is prepared by the method described in the third aspect of the invention.

[0076] In another preferred embodiment, the EnvA pseudotype CVS N2c rabies virus is used as a reagent for preparing reverse "cross-monosynaptic" tracing of neurons.

[0077] 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

[0078] Figure 1 The target gene plasmid snapgene map is displayed.

[0079] Figure 2 This diagram illustrates the sorting of N2a-EnvA cells. The X-axis represents the EGFP fluorescence channel value, indicating the cell's green fluorescence expression level; the Y-axis represents the FSC forward scattering channel value, indicating the cell size. Each dot in the diagram represents one cell.

[0080] Figure 3 The image shows the expression of EnvA in N2a-EnvA cells. The image is an image of N2a-EnvA cells before sorting under a fluorescence microscope; scale bar 200 μm; left: bright field, right: EGFP.

[0081] Figure 4Growth curves of CVS-N2cΔG-EGFP (EnvA) are shown. CVS-N2cΔG-EGFP producing an EnvA capsid was packaged in N2a-EnvA-1 and N2a-EnvA-2 cell lines. Viral supernatants were harvested 3–11 days post-inoculation (sup1–sup9), and the titer of each supernatant was calculated; unit: iu / mL.

[0082] Figure 5 The images show mCherry expression in CVS-N2cΔG-mCherry (EnvA) under a fluorescence microscope. The images are taken on day 3 and day 5 after inoculation at MOI=2, scale bar 200 μm. Left: EGFP, expressed by the N2a-EnvA-1 cell line; Right: mCherry, expressed by the CVS-N2cΔG-mCherry virus.

[0083] Figure 6 The histogram shows the qPCR titer results of concentrated viruses prepared from the N2a-EnvA cell line. The figure displays the titer results of CVS-N2cΔG-mcherry and three other CVS-N2cΔG viruses concentrated using the N2a-EnvA cell line, obtained through three parallel qPCR tests. One-way ANOVA was used to analyze the titer results for each virus; p>0.05, indicating no significant difference and the data were relatively parallel. Detailed Implementation

[0084] Through extensive and in-depth research, the inventors have, for the first time, constructed a cell line for the efficient propagation of EnvA pseudotyped CVSN2c rabies virus. This cell line is a polyclonal cell line, with its genome integrated with exogenous expression cassettes expressing EnvA and fluorescent reporter genes, which can be used to encapsulate CVS N2c rabies virus that produces an EnvA coat. The supernatant titer of the EnvA pseudotyped rabies virus packaged in this cell line is consistently above 1E6 IU / mL, an order of magnitude higher than existing toxin-producing cell lines. Therefore, it can efficiently propagate EnvA pseudotyped CVS N2c rabies virus for reverse trans-monosynaptic labeling and tracking of specific types of neurons expressing the TVA protein.

[0085] Based on this, the present invention was completed.

[0086] the term

[0087] 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.

[0088] 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.).

[0089] 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”.

[0090] SAD B19 virus strain and CVS N2c virus strain

[0091] Both SAD B19 and CVS N2c virus strains are RV virus strains. CVS N2c virus strain is a mutant strain established by Reardon et al. in 2016, which has both advantages and disadvantages compared to SAD B19 virus strain.

[0092] Advantages of CVS N2c virus strain: CVS N2c virus strain has lower neurotoxicity, and infected neurons have better survival ability, making it more suitable for experiments that require long-term observation; CVS N2c virus strain has stronger reverse labeling efficiency; CVS N2c virus strain does not infect astrocytes.

[0093] Disadvantages of CVS N2c virus strain: The rescue process for CVS N2c virus strain is more difficult and takes longer than that for SAD B19 virus strain.

[0094] In order to improve the preparation efficiency of CVS N2c virus strains and rapidly obtain high-titer viruses, this application provides a cell line that can efficiently proliferate EnvA pseudotype CVS N2c rabies virus.

[0095] EnvA pseudotype CVS N2c rabies virus

[0096] As used herein, the terms "EnvA pseudotyped CVS N2c rabies virus," "CVSN2c rabies virus with G protein deficiency in the EnvA shell," "CVS N2c rabies virus with G protein deficiency coated by EnvA," and "CVS N2cΔG(EnvA)" are used interchangeably and all refer to recombinant CVS N2c rabies virus with a G protein deficiency in its viral genome, coated by an EnvA shell. In one specific embodiment of the present invention, the above terms refer to the recombinant rabies virus CVS-N2cΔG-mCherry(EnvA) prepared in the examples, which is produced by packaging using the N2a-EnvA cell line of the present invention, replacing the B19G shell of the CVS-N2cΔG-mCherry(B19G) recombinant rabies virus with an EnvA shell. The EnvA pseudotyped CVS N2c rabies virus produced by the present invention can be used to label neurons expressing TVA, achieving reverse cross-monosynaptic tracing of neurons.

[0097] As used herein, the terms "CVS N2c rabies virus with G protein deletion" and "CVS N2c-ΔG" are used interchangeably, both referring to recombinant CVS N2c rabies virus in which the glycoprotein gene G, responsible for viral invasion, has been deleted from the genome. As used herein, the term "CVS-N2cΔG-mCherry(B19G)" refers to a recombinant CVS N2c rabies virus in which the mCherry red fluorescent protein gene has replaced the glycoprotein G gene in the CVS N2c rabies virus genome, but the original glycoprotein B19G is used for virus packaging, and the viral coat remains the B19G glycoprotein.

[0098] Cell lines for efficient propagation of EnvA pseudotype CVS N2c rabies virus

[0099] In one aspect of the invention, a cell line for the efficient propagation of EnvA pseudotyped CVS N2c rabies virus is provided, said cell line being an N2a 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 N2a-EnvA-1 cell line, whose genome integrates an exogenous expression cassette expressing EnvA and EGFP.

[0100] The N2a-EnvA cell line of this invention is a polyclonal cell line, a group of cells expressing high-intensity 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 N2a-EnvA cell line of this invention can be used for efficient propagation of EnvA pseudotyped CVS N2c rabies virus, and the supernatant titer of the packaged EnvA pseudotyped CVS N2c rabies virus is consistently above 1E6 IU / mL.

[0101] As used in this invention, the terms "cell line of the present invention", "cell line for efficient propagation of EnvA pseudotype CVS N2c rabies virus", "cell line as described in this invention", "cell line for efficient propagation of EnvA pseudotype CVS N2c rabies virus of the present invention", and "cell line for efficient propagation of EnvA pseudotype CVS N2c rabies virus as described in this invention" are used interchangeably and all refer to the N2a cell line as described in the first aspect of this invention.

[0102] Methods for constructing cell lines for efficient propagation of EnvA pseudotype CVS N2c rabies virus

[0103] In another aspect of the invention, a method for preparing a cell line for efficient proliferation of EnvA pseudotype CVS N2c rabies virus as described herein is also provided, the method comprising the following steps:

[0104] (a) Preparation of viral vectors containing exogenous expression cassettes expressing EnvA and fluorescent reporter genes;

[0105] Specifically, it includes the following steps:

[0106] (a1) Construct a target gene plasmid containing an exogenous expression cassette expressing EnvA and a fluorescent reporter gene; preferably, the target gene plasmid is an exogenous expression cassette containing EnvA and EGFP genes.

[0107] The expression cassette plasmid EnvA-IRES-EGFP; and

[0108] (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;

[0109] 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.

[0110] (b) Infect N2a cells with the viral vector obtained from step (a) to obtain infected N2a cells;

[0111] Specifically, based on an infection multiplicity (MOI) of 800–1500, preferably 1000–1200, and more preferably about 1000, N2a cells are inoculated into 96-well plates, wherein the number of N2a cells per well is 4–8K cells, preferably 5–6K cells, and more preferably 5.5K cells per well; and

[0112] (c) Passage and expand the infected N2a cells from step (b), and then use flow cytometry to screen and obtain N2a cell populations expressing moderate fluorescence intensity, which is the cell line used for efficient proliferation of EnvA pseudorabies virus.

[0113] Specifically, 24-48 hours post-infection, the infected N2a cells were passaged and expanded, i.e., the infected N2a cells in 96-well plates were expanded into 24-well plates; once the cells had grown into a confluent monolayer in the 24-well plates (i.e., the cell confluence reached more than 90%), the cells were expanded into 10 cm culture dishes for further culture; once the infected N2a cells had grown into a confluent monolayer in the 10 cm culture dishes (i.e., the cell confluence reached more than 90%), screening began;

[0114] The flow cytometry sorting process involves separating fluorescently positive N2a 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, comprising the top 1.5%-2% (ideally, the top 1.2%-1.5%, more preferably, the top 1.1%-1.2%, and best, the top 1.05%) of the entire cell collection. The medium-intensity fluorescence group consists of cells with the second highest fluorescence intensity, comprising the top 3%-5% of the entire cell collection. The cell collection in the high-intensity fluorescence group is the target cell line.

[0115] Packaging EnvA pseudorabies virus

[0116] This invention also provides a method for packaging EnvA pseudotyped CVS N2c rabies virus, using the cell line described in this invention. Specifically, the method includes the following steps:

[0117] (S1) Infect the cell line as described in this invention with G protein-deficient CVS N2c rabies virus (CVS N2cΔG); and

[0118] (S2) Culture the infected cell line to obtain EnvA pseudotype CVS N2c rabies virus.

[0119] In one specific embodiment of the present invention, the method includes: infecting the N2a-EnvA cell line of the present invention with CVS-N2cΔG-mCherry (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; collecting the cell culture supernatant from day 3 to day 5 after infection, thereby obtaining the EnvA pseudotype CVS N2c rabies virus CVS-N2cΔG-mCherry (EnvA).

[0120] In another specific embodiment of the present invention, the method includes: infecting the N2a-EnvA cell line of the present invention with CVS-N2cΔG-1 / 2 / 3; 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; collecting the cell culture supernatant from day 3 to day 5 after infection to obtain EnvA-coated CVS-N2cΔG-1 / 2 / 3.

[0121] application

[0122] The present invention also provides the use of the cell line as described herein for packaging EnvA pseudotype CVS N2c rabies virus, the resulting EnvA pseudotype rabies virus being used to prepare reagents for reverse "cross-monosynaptic" tracing of neurons.

[0123] The sequence information involved in this invention:

[0124] EnvA nucleotide sequence (SEQ ID NO:1)

[0125]

[0126] EnvA amino acid sequence (SEQ ID NO: 2)

[0127] MEAVIKAFLTGHPGKVSKKDSKKKPPATSKKDPEKTPLLPTRVNYIIGVLVLCEVTGVRADVHLLEQPGNLWITWANRTGQTDFCLSTQSATSPFQTCLIGIPSPISEGDFKGYVSDNCTTLEPHRLVSRGIPGGPENSTTLTYQKVSCLLLKLNVSLLDEPSELQLLGSQSLPNITNITRIPSVAGGCIGFTPYDSPAGVYGWDRREVTHILLTDPGNNPFFDKASNSSKPFTVVTADRHNLFMGSEYCGAYGYRFWEMYNCSQMRQNWSICQDVWGRGPPENWCTSTGGTWVNQSKEFNETAPFSFTVNCTGSNLGNVSGCCGEPITILPPEAWVDSTQGSFTKPKALPPAIFLICGDRAWQGIPSRPVGGPCYLGKLTMLAPKHTDILKVLVNSSRTGIRRKRSTSHLDDTCSDEVQLWGPTARIFASILAPGVAAAQALREIERLACWSVKQANLTTSLLGDLLDDVTSIRHAVLQNRAAIDFLLLAHGHGCEDVAGMCCFNLSDHSESIQKKFQLMKEHVNKIGVDSDPIGSWLRGLFGGIGEWAVHLLKGLLLGLVVILLLVVCLPCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGETRL*

[0128] EGFP nucleotide sequence (SEQ ID NO: 3)

[0129] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0130] Amino acid sequence of EGFP (SEQ ID NO: 4)

[0131] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0132] The main advantages of this invention include:

[0133] (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.

[0134] (2) The cell line of the present invention can efficiently proliferate EnvA pseudotype CVS N2c rabies virus. Compared with the prior art (the virus production titer is at the level of 10 to the power of 5 iu / mL), the supernatant titer of EnvA pseudotype rabies virus produced in the cell line of the present invention is stable at more than 1E6 iu / mL, which is an order of magnitude higher.

[0135] (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 pseudotype CVS N2c rabies virus.

[0136] (4) The EnvA pseudotype CVS N2c 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; and due to its high titer, it can trace more brain circuit information.

[0137] 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.

[0138] Example 1: Preparation of N2a-EnvA cell line

[0139] 1.1 Virus Packaging Plasmid

[0140] 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.

[0141] 1.2 Cells

[0142] Both HEK-293T cells and N2a cells were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0143] 1.3 Experimental Procedure

[0144] (1) Preparation of EnvA-expressing retroviruses:

[0145] 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.

[0146] b) One day before transfection, seed approximately 80% confluent HEK-293T cells into culture dishes coated with PLL the day before, about 1.8E7 / dish.

[0147] c) On the day of transfection, confirm that the cells have reached 70-80% confluency, and use DNA:L2K (Lipofectamine) TM 2000 transfection reagent (Invitrogen, catalog number: 11668019) was used at a ratio of 1:2.3. Endotoxin-free plasmids (in one 15cm culture dish: target plasmid EnvA-IRES-GFP 22.5ug, plasmid gag / pol 16.9ug, pCAG-VSVG 5.6ug) were used to transfect 293T cells. During DNA and L2K incubation, the culture medium in the 15cm dish was replaced with 12ml of Opti- Medium

[0148] (Serious-reduced medium, Gibco, catalog number: 11058021), 5-6 hours after 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);

[0149] d) Harvest the supernatant three days after transfection. Centrifuge at 2000 rpm for 5 minutes, then use 0.45-

[0150] After filtration through a μm Stericup filter (BIOFIL, catalog number: FCF000007), the virus was concentrated by high-speed centrifugation (22000 rpm, 4°C for 2 hours) to obtain concentrated virus.

[0151] (2) Preparation of cell lines: The concentrated virus was inoculated into 96-well plates with approximately 5.5K N2a cells (200 μL of medium per well) at an MOI of 1000. After 24 hours, the fluorescence was observed and the medium was changed. 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.

[0152] (3) Cell line sorting: N2a-EnvA cell lines were screened by flow cytometry.

[0153] The specific method of flow sorting is as follows:

[0154] 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.

[0155] b) Centrifugation: After resuspending the cells, centrifuge at 200g for 5 minutes at room temperature;

[0156] 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.

[0157] d) Centrifugation: After incubation, add 5 ml of DMEM to resuspend the cells and centrifuge at 200 g for 5 min at room temperature;

[0158] e) Sorting: After centrifugation, discard the supernatant, resuspend the cells in 1 ml of DMEM, and pass them through the pore size...

[0159] 35µm flow cytometer ( 12x75mm test tubes with cell sieve caps, part number: 352235

[0160] After filtration, a single-cell suspension was prepared and directly subjected to flow cytometry sorting. Two cell collection groups were plotted based on fluorescence intensity: a high-intensity fluorescence group (top 1.05%) and a medium-intensity fluorescence group (top 2.93%). The final sorted positive cells were named N2a-EnvA-1 / 2 (see figure).

[0161] 2. P1 represents N2a-EnvA-1 cells comprising the top 1.05% of the green fluorescent cells; P2 represents N2a-EnvA-2 cells comprising the top 2.93% of the green fluorescent cells. Cells were passaged and amplified for preservation after reaching a confluent monolayer. The aforementioned percentages refer to the percentages of the entire sorted cell population.

[0162] 1.4 Results and Conclusions

[0163] Green fluorescence expression was observed in cells infected with the virus, indicating that the EnvA gene had been successfully integrated into N2a cells (see...). Figure 3 However, the fluorescence intensity varies among different cells.

[0164] 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.

[0165] Therefore, the cell lines were sorted by flow cytometry, and a green fluorescent positive N2a cell line with stable expression of EGFP and EnvA was obtained. This line was named N2a-EnvA-1 / 2 and was used for subsequent virus preparation tests.

[0166] Example 2: Validation of the packaging efficiency of N2a-EnvA cell line for EnvA pseudotyped CVS N2c rabies virus

[0167] 2.1 Experimental Materials

[0168] Cell lines: N2a-EnvA-1 and N2a-EnvA-2 cell lines obtained in Example 1.

[0169] Virus: CVS-N2cΔG-EGFP(B19G), 4.53E6 iu / mL.

[0170] 2.2 Experimental Procedure

[0171] (1) Proliferation of CVS-N2c virus with EnvA outer shell:

[0172] 1) One day before inoculation, N2a-EnvA-1 cells with good growth status at 600K were passaged into two 6-well plates, and N2a-EnvA-2 cells with good growth status at 600K were passaged into two other 6-well plates.

[0173] 2) On the day of inoculation, count one well for each of the two cell types, and inoculate another well with CVS-N2cΔG-EGFP(B19G) (virus titer 4.53E6 iu / mL) according to MOI=2.

[0174] 3) 24 hours after inoculation, wash the cells twice with 1 ml of DPBS, and then add 2 ml of DMEM containing 10% fetal bovine serum to continue culturing.

[0175] 4) 48 hours after inoculation, wash the cells twice with 1 ml of DPBS, and then add 2 ml of DMEM containing 10% fetal bovine serum to continue culturing.

[0176] 5) From day 3 to day 11, collect the supernatant daily. Add 2 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℃.

[0177] (2) Determination of CVS virus supernatant titer in proliferated EnvA capsid using FACS: The supernatant was seeded into 293T-TVA950-BFP cells (a cell line expressing the EnvA-specific receptor TVA950), and the virus titer was determined using FACS. The specific steps of this method are as follows:

[0178] a) One day before inoculation, seed 293T-TVA950-BFP cells in 96-well plates at 30K / well.

[0179] b) On the day of receiving the virus, dilute the supernatant according to Table 1 below:

[0180] Table 1

[0181] 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

[0182] 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.

[0183] 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.

[0184] 2.3 Results

[0185] In this experiment, supernatant harvesting began on day 3 and was carried out once a day until day 11 after inoculation, with a total of 9 supernatants (sup1-sup9) collected.

[0186] The viral titer of the daily harvested supernatant was determined (see Table 2), and a viral growth curve was plotted (see Table 2). Figure 4 Based on the viral growth curve, the N2a-EnvA cell line's ability to package the virus is mainly concentrated on days 3-6, i.e., sup1-sup4. Therefore, when performing EnvA recoating experiments on the virus, collecting the supernatant from days 3-5 (sup1-sup3) will yield a sufficient viral load. Furthermore, the N2a-EnvA-1 cell line demonstrates a superior ability to proliferate CVS N2c virus compared to the N2a-EnvA-2 cell line.

[0187] Table 2. Viral titers in supernatant for N2a-EnvA cell lines.

[0188]

[0189]

[0190] 2.4 Verification Experiment

[0191] To further confirm the CVS N2c virus proliferation ability of the N2a-EnvA-1 cell line, CVS-N2cΔG-mCherry (EnvA) was subsequently packaged on N2a-EnvA-1 cells. Fluorescent expression after inoculation showed good mCherry expression of CVS-N2cΔG-mCherry on days 3 and 5 post-inoculation (see [link to data]). Figure 5 Viral supernatants were harvested 3-5 days after inoculation (sup1-sup3), and the titer of each supernatant was calculated (see Table 3). After concentrating the viral supernatants, the titer was measured three times, and the viral titer remained stable above the 10th power in each case (see Table 4).

[0192] Table 3. Supernatant titers of CVS-N2cΔG-mCherry (EnvA)

[0193] Shangqing Viral titer (iU / mL) Sup1-D3 1.02E+07 Sup2-D4 9.27E+06 Sup3-D5 5.34E+06

[0194] In addition to CVS-N2cΔG-mCherry, the N2a-EnvA-1 cell line of this invention was subsequently used for inoculation at MOI=2 to repeatedly package three different CVS-N2cΔG(EnvA) viruses, represented here as CVS-N2cΔG-1, CVS-N2cΔG-2, and CVS-N2cΔG-3. Since the reporter gene cannot be used to determine viral titers by flow cytometry, SYBR-Green (which can bind to the grooves of double-stranded DNA and emit green fluorescence, enabling real-time quantitative PCR amplification) was used to determine the concentrated viral titers using qPCR (real-time quantitative PCR). Simultaneously, the viral titers of the concentrated CVS-N2cΔG-mCherry were measured as a control. Specific titer results are shown in Table 4, and statistical graphs are shown below. Figure 6 .

[0195] Table 4. Statistical table of qPCR titers of concentrated virus prepared from N2a-EnvA cell line.

[0196] Titration (iu / mL) Repeat-1 Repeat-2 Repeat-3 CVS-N2cΔG-mCherry 1.11E+10 1.09E+10 1.03E+10 CVS-N2cΔG-1 3.33E+10 4.55E+10 6.28E+10 CVS-N2cΔG-2 1.34E+10 1.6E+10 1.56E+10 CVS-N2cΔG-3 4.37E+10 4.17E+10 3.79E+10

[0197] Comparing the concentrated virus titers of several groups reveals that the concentrated CVS-N2cΔG prepared by the N2a-EnvA-1 cell line can reach E10 and above, which further verifies that this cell line can be used for stable and efficient propagation of EnvA pseudotype CVS N2c rabies virus.

[0198] 2.5 Conclusion

[0199] The N2a-EnvA cell line of the present invention can efficiently proliferate EnvA pseudotype CVS N2c rabies virus, effectively replacing the outer shell of CVS-N2cΔG virus from B19G to EnvA; and the titers (iu / mL) of the first three harvested supernatants can be consistently above the 6th power, and the virus titers after concentration can reach above the 10th power.

[0200] 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 efficient propagation of EnvA pseudotype CVS N2c rabies virus, characterized in that, The cell line is the N2a 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 of claim 1, wherein, The cell line used for efficient propagation of EnvA pseudotype CVS N2c rabies virus is a polyclonal cell line.

3. The cell line of claim 1, wherein, The fluorescent reporter genes include EGFP, GFP, BFP, mCherry, tdTomato, or combinations thereof.

4. The cell line of claim 1, wherein, 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 high-intensity fluorescence, wherein "high-intensity fluorescence" means that the fluorescence intensity accounts for the top 1.5%-2% of the total fluorescence intensity of the sorted cell population, preferably the top 1.2%-1.5%, more preferably the top 1.1%-1.2%, and most preferably the top 1.05%.

6. A method for preparing a cell line for efficient propagation of EnvA pseudotype CVS N2c 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 N2a cells with the viral vector obtained from step (a) to obtain infected N2a cells; and (c) The infected N2A cells from step (b) are passaged and expanded, and then flow cytometry is used to screen and obtain a population of N2a cells expressing high fluorescence intensity, which is the cell line used for efficient proliferation of EnvA pseudotype CVS N2c rabies virus.

7. The method as described in claim 6, characterized in that, Based on fluorescence intensity, the fluorescently positive N2a cells detected by the instrument were sorted into two cell collection groups: 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 the top 1.5%-2% of the entire sorted cell population (preferably, the top 1.2%-1.5%, more preferably, the top 1.1%-1.2%, and best, the top 1.05%). The medium-intensity fluorescence group refers to the cells with the second highest fluorescence intensity, accounting for 3%-5% of the entire sorted cell population. The cell population in the high-intensity fluorescence group is the target cell line.

8. A method for packaging EnvA pseudotype CVS N2c 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 CVS N2c rabies virus (CVS N2cΔG); and (S2) Culture the infected cell line to obtain EnvA pseudotype CVS N2c rabies virus.

10. Use of the cell line according to any one of claims 1-5 for packaging EnvA pseudotype CVS N2c rabies virus, thereby for preparing a reagent for reverse "across monosynaptic" tracing of neurons.

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