Cell line for saving rabies virus and application thereof

Abstract

The invention relates to a cell line for saving rabies virus and application thereof. A cell comprising, expressing and / or secreting B19G or a variant having at least 80% sequence identity thereto and retaining function is provided. According to the cell line, viruses can be efficiently rescued, and passage of the viruses is reduced.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically to cell lines for rescuing rabies virus and their applications. Background Technology

[0002] The human brain contains at least one hundred billion neurons of various types. It is estimated that each neuron has 1,000-15,000 synapses, meaning the human brain contains an average of over one trillion synaptic connections. These connections form a complex neural network, which is the foundation for the brain's specific functions. Currently, the pathogenic mechanisms of normal physiological activities and brain diseases are unclear, partly due to a lack of information about the connections within the brain's neural networks. Therefore, conducting research on brain neural circuits and mapping high-precision brain functional connectivity is crucial for addressing these issues. 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 for neuroscientists. Wild-type rabies virus belongs to the genus Lyssavirus in the family Rhabdoviridae. It has an envelope and its genome is single-stranded negative-sense RNA. The virus particle is approximately 250 nm long and 70 nm in diameter. 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. Currently, the commonly used vector in neuroscience research is based on the rabies virus vaccine strain SAD B19. Through reverse genetics, the glycoprotein gene G, responsible for viral invasion, has been deleted from the genome, resulting in the first-generation rabies virus (ΔG). Traditional methods use BHK cells, which have low transfection efficiency, leading to low virus rescue efficiency and thus low viral titers. The virus needs to be passaged multiple times to reach the ideal titer, but rabies virus undergoes many mutations during multiple passages, causing some viruses to lose their replication function, which reduces the labeling efficiency in animal experiments. More efficient cell lines and methods for the rescue of first-generation rabies virus are still needed in this field. Summary of the Invention

[0004] The first aspect of this invention provides a retroviral vector, comprising:

[0005] (1) A nucleic acid sequence encoding B19G, wherein the amino acid sequence of B19G is as shown in SEQ ID NO:2 or has at least 80% sequence identity with it, and / or

[0006] (2) A nucleic acid sequence having at least 80% sequence identity with (1) and encoding B19G as shown in SEQ ID NO:2, and / or

[0007] (3)(1) or (2) reverse complementary sequences.

[0008] In one or more embodiments, the retroviral vector is derived from Moloney murine leukemia virus, preferably, the retroviral vector is GenBank:AF033811.1.

[0009] In one or more embodiments, the nucleic acid sequence of the B19G is shown in SEQ ID NO:1.

[0010] This invention provides a retrovirus, comprising:

[0011] (1) The nucleic acid sequence of B19G, wherein the amino acid sequence of B19G is as shown in SEQ ID NO:2 or has at least 80% sequence identity with it, and / or

[0012] (2) A nucleic acid sequence having at least 80% sequence identity with (1) and encoding B19G as shown in SEQ ID NO:2, and / or

[0013] (3)(1) or (2) reverse complementary sequences.

[0014] In one or more embodiments, the retrovirus comprises the retroviral vector described in the first aspect of this document.

[0015] In one or more embodiments, the retrovirus further comprises a nucleic acid construct having the genes required for the retrovirus.

[0016] In one or more embodiments, the required genes for the retrovirus include one or more selected from the following: gag, pol, VSVG.

[0017] This invention provides a cell, wherein:

[0018] (1) Contains, expresses and / or secretes B19G or a variant that has at least 80% sequence identity with B19G and retains its function.

[0019] (2) Contains the following sequences: i) a nucleic acid sequence of B19G, wherein the amino acid sequence of B19G is as shown in SEQ ID NO:2, and / or, ii) a sequence having at least 80% sequence identity with i) and encoding B19G as shown in SEQ ID NO:2, and / or, iii) the reverse complementary sequence of i) or ii).

[0020] (3) A nucleic acid construct containing the sequence described in (2),

[0021] (4) Contains the retrovirus described in any of the embodiments herein.

[0022] In one or more embodiments, the cells are used to prepare rabies virus. In one or more embodiments, the cells are used to prepare rabies virus (RV). In one or more embodiments, the rabies virus is a rabies virus lacking the G protein (glycoprotein).

[0023] In one or more embodiments, the nucleic acid construct is a vector.

[0024] In one or more embodiments, the vector is a cloning vector, an integration vector, or an expression vector.

[0025] In one or more embodiments, the vector is a retroviral vector.

[0026] In one or more embodiments, the cells are HEK293 cells, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0027] The present invention provides a method for preparing a cell line stably expressing B19G, the method comprising: introducing a nucleic acid construct containing a nucleic acid sequence encoding B19G into the cell, wherein the nucleic acid sequence is operatively linked to a promoter, and the amino acid sequence of B19G is shown in SEQ ID NO:2.

[0028] In one or more embodiments, the method includes:

[0029] (1) Obtain the retroviral vector described in the first aspect of the present invention or the retrovirus described in the second aspect of the present invention.

[0030] (2) Inoculate the retroviral vector or the retrovirus into the cells.

[0031] (3) Screening for cells that stably express B19G.

[0032] In one or more embodiments, the cells are HEK293 cells. HEK293 cells include HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0033] In one or more embodiments, the cells are 293T cells that have been revived and cultured for more than three generations.

[0034] In one or more embodiments, the nucleotide sequence of the B19G is shown in SEQ ID NO:1.

[0035] In one or more embodiments, in (2) the virus is inoculated into the cells at an MOI of 1-1000, preferably MOI of 100.

[0036] A fourth aspect of the present invention provides a cell line prepared by the method described in the third aspect of the present invention.

[0037] The present invention also provides a method for preparing or rescuing a virus, comprising preparing the virus using the cells or cell lines described in any embodiment herein.

[0038] In one or more embodiments, the virus is a rabies virus (RV). In one or more embodiments, the rabies virus is a rabies virus lacking the G protein (glycoprotein).

[0039] In one or more embodiments, the method includes:

[0040] (1) The RV genome is introduced into the cells or cell lines containing the following proteins: rabies virus nucleoprotein (N protein), rabies virus phosphoprotein (P protein), rabies virus glycoprotein (G protein), rabies virus macroprotein (L protein), and optionally, RNA polymerase (e.g., T7 RNA polymerase).

[0041] (2) Incubate the cells or cell lines under conditions suitable for the production of rabies virus.

[0042] In one or more embodiments, the method includes:

[0043] (1) The RV genome is introduced into the cells or cell lines in which the following proteins are expressed: rabies virus nucleoprotein, rabies virus phosphoprotein, rabies virus glycoprotein, and rabies virus macroprotein. Optionally, RNA polymerase (e.g., T7 RNA polymerase) may also be expressed in the cell lines.

[0044] (2) Incubate the cells or cell lines under conditions suitable for the production of rabies virus.

[0045] In one or more embodiments, the method includes,

[0046] (1) Introduce the following genes or genomes into the cells or cell lines: RV genome, rabies virus nucleoprotein gene, rabies virus phosphoprotein gene, rabies virus glycoprotein gene, rabies virus macroprotein gene, and optionally RNA polymerase gene, and

[0047] (2) Incubate the cells or cell lines under conditions suitable for the production of rabies virus.

[0048] In one or more embodiments, the method includes,

[0049] (1) Introduce the following nucleic acid constructs into the cells or cell lines: vectors containing the RV genome, vectors expressing rabies virus nucleoproteins, vectors expressing rabies virus phosphoproteins, vectors expressing rabies virus glycoproteins, vectors expressing rabies virus macroproteins, and optionally vectors expressing RNA polymerase.

[0050] (2) Incubate the cells or cell lines under conditions suitable for the production of rabies virus.

[0051] In one or more embodiments, the introduction is achieved by transfection. In one or more embodiments, the transfection reagent is PEI or Lipofectamine 2000, preferably, the transfection is performed using PEI.

[0052] In one or more embodiments, transfection includes mixing a nucleic acid construct (e.g., a vector) containing the gene or genome with a transfection reagent (e.g., PEI) and then mixing the mixture with cells. Preferably, the weight ratio of the nucleic acid construct to the transfection reagent (e.g., PEI) is from 10:1 to 1:10, more preferably 1:3.

[0053] In one or more embodiments, the RV genome may or may not contain G protein gene sequences. In one or more embodiments, the G protein gene sequences in the RV genome are replaced with fluorescent protein (e.g., mCherry) gene sequences. Preferably, the sequence of the RV genome is as shown in SEQ ID NO:4.

[0054] In one or more embodiments, the amino acid sequence of the N protein is shown in SEQ ID NO:5.

[0055] In one or more embodiments, the amino acid sequence of the P protein is shown in SEQ ID NO:6.

[0056] In one or more embodiments, the amino acid sequence of the G protein is shown in SEQ ID NO:7.

[0057] In one or more embodiments, the amino acid sequence of the L protein is shown in SEQ ID NO:8.

[0058] In one or more embodiments, the amino acid sequence of the T7 RNA polymerase is shown in SEQ ID NO:9.

[0059] The present invention also provides the use of the retroviral vector, retrovirus, cell and / or cell line described in any embodiment of the present invention in the preparation of viruses, increasing virus titers, and improving virus packaging efficiency.

[0060] In one or more embodiments, the virus is a rabies virus (RV). In one or more embodiments, the rabies virus is a rabies virus lacking the G protein (glycoprotein).

[0061] The present invention also provides a kit comprising a retroviral vector, a retrovirus, a cell, or a cell line as described in any embodiment of the present invention.

[0062] The present invention also provides the use of the retroviral vector, retrovirus, cell, or cell line described in any embodiment herein in the preparation of reagents for increasing viral titers or improving viral packaging efficiency.

[0063] In one or more embodiments, the virus is a rabies virus (RV). In one or more embodiments, the rabies virus is a rabies virus lacking the G protein (glycoprotein). Attached Figure Description

[0064] Figure 1 The expression of the virus in 293T cells. The retrovirus was used to infect 293T cells at MOI=100. The image shows 293T-B19G cells before sorting under a fluorescence microscope. Scale bar: 200 μm. Left: Bright field. Right: EGFP.

[0065] Figure 2 Cell line expression after sorting. Cell lines exhibiting stable fluorescence expression were sorted by flow cytometry and named 293T-B19G. Scale bar 200 μm. Left: Bright field. Right: EGFP.

[0066] Figure 3mCherry expression in RVΔG-4mCherry under a fluorescence microscope. Images are taken on days 3, 6, and 9 post-transfection. Scale bar: 200 μm. Left: Bright field or EGFP, EGFP expressed by the 293T-B19G cell line. Right: mCherry, expressed by RVΔG-4mCherry virus.

[0067] Figure 4 RVΔG-4mCherry growth curves. RV cells expressing mCherry were packaged in 293T cells (with or without B19G expression). Viral supernatants were harvested 3–10 days post-transfection, and the titer of each supernatant was calculated. The blue line represents the viral titer produced by RVΔG-4mCherry packaged in 293T cells over 8 consecutive days. The green line represents the viral titer produced by RVΔG-4mCherry packaged in 293T-B19G cells over 8 consecutive days. Unit: IU / mL.

[0068] Figure 5 RVΔG-4mCherry growth curves. Viral supernatants were harvested 3–10 days post-transfection, and the titer of each supernatant was calculated. The green line represents the viral titer produced by RVΔG-4mCherry packaged for 8 consecutive days during cell line testing. The red line represents the supernatant titer from 5 independent replicates, expressed as mean and standard deviation. Unit: iu / mL. Detailed Implementation

[0069] This invention is the first to discover that constructing 293T cells stably expressing B19G can significantly increase viral titer and viral packaging efficiency. It has broad application prospects in the field of neural circuit tracing.

[0070] Nucleic acid constructs, viruses and cells

[0071] A first aspect of this invention provides a nucleic acid construct comprising the coding sequence of B19G. In this document, the nucleotide sequence of said B19G has the variant shown in SEQ ID NO:1 or having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity with it. The sequence identity described in this invention can be measured using sequence analysis software, such as a computer program BLAST with default parameters, like BLASTP or BLASTN.

[0072] The full-length nucleic acid sequences or fragments thereof of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. Once the relevant sequences are obtained, they can be obtained in large quantities using recombination. This typically involves cloning them into a vector, transforming them into cells, and then isolating the relevant sequences from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0073] The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, techniques described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley), or other standard textbooks. Alternatively, the polynucleotide and vector can be reconstituted into liposomes for delivery to target cells. Vectors containing the polynucleotides of the present invention can be transferred into host cells using well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0074] The present invention also relates to polynucleotides that hybridize with the above-described nucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0075] The nucleic acid constructs described herein can be expression cassettes, which, depending on the expression scenario, may consist of a promoter and a B19G coding sequence. In some implementations, the expression cassette may also include a 5' ITR and a 3' ITR. The promoters include, but are not limited to, the CAG promoter, the CMV promoter, and the H1 promoter.

[0076] In some implementations, the nucleic acid construct is a vector. The vector can be a plasmid, granule, virus, or viral vector. Vectors include cloning vectors, integration vectors, and expression vectors. In addition to the nucleotide molecules described in this invention, expression vectors typically contain other elements commonly found in vectors, such as multiple cloning sites, resistance genes, and replication initiation sites. Vectors that can effectively express proteins in the human body, especially in 293T cells, are preferred for use in this invention, including but not limited to retroviral vectors, lentiviral vectors, and adenoviral vectors.

[0077] Expression vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to as "flanking sequences" in some embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a leader sequence encoding a polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting a nucleic acid encoding the protein to be expressed, and optional marker elements. Exemplary expression vectors include pCAG, a retroviral vector shown in GenBank: AF033811.1.

[0078] The vector may optionally contain a "tag" encoding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the fusion polypeptide; the oligonucleotide sequence encodes a polyhistidine (such as 6His) or another "tag," such as FLAG, HA, or myc. This tag typically fuses with the polypeptide during expression and can serve as a means of affinity purification or detection of the protein from host cells. Affinity purification can be performed, for example, by column chromatography using an antibody against this tag as an affinity matrix. The tag may optionally be subsequently removed from the purified protein by various methods, such as using certain peptidases for lysis.

[0079] Flanking sequences can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), heterozygous (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Similarly, the source of the flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence functions in a host cell mechanism and can be activated by a host cell mechanism.

[0080] In this paper, the virus described is a retrovirus whose genome includes three genes: the gag gene (encoding the core protein of the virus), the pol gene (encoding reverse transcriptase), and the env gene (encoding the surface glycoprotein of the virus).

[0081] This invention also provides cell lines comprising the nucleic acid constructs or retroviruses described in any of the embodiments herein. The cells are preferably HEK 293T cells.

[0082] Methods and uses

[0083] The present invention also provides a method for preparing the cells, the method comprising: (1) preparing the retrovirus as described in the second aspect of the present invention, (2) inoculating the retrovirus prepared in (1) into cells, and (3) sorting and screening the cells in (2). Preferably, the inoculation in (2) is to inoculate the virus into 293T cells at an MOI of 100.

[0084] The present invention also provides the use of the nucleic acid constructs, retroviruses, and cell lines described in any embodiment of the present invention in packaging viruses, the uses including increasing virus titer and increasing virus packaging efficiency.

[0085] In one or more embodiments, the virus is rabies virus. This invention also provides the use of the nucleic acid constructs, retroviruses, and cell lines described in any embodiment herein in the preparation of reagents for increasing viral titers and improving viral packaging efficiency. Exemplarily, this invention uses 293T cells to prepare the virus. Virus transfection methods suitable for 293T cells are well known in the art, such as PEI or Lipofectamine 2000. Preferably, when the transfection reagent (e.g., PEI) is toxic to 293T cells, viral production can be accelerated by accelerating cell death.

[0086] Reagent test kit

[0087] The present invention also provides a kit comprising the nucleic acid construct, retrovirus, or cell line described in any embodiment of the present invention.

[0088] In some embodiments, the kit also includes instructions for use. Specifically, such a kit may include one or more reagents described herein, along with instructions describing the intended use and proper use of these reagents. In some embodiments, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing samples and administering them to a subject. In some embodiments, the reagents in the kit are pharmaceutical formulations and dosages suitable for a particular application and method of reagent administration. Kits used for research purposes may contain appropriate concentrations or amounts of components for performing various experiments.

[0089] The kit may be designed to facilitate use of the methods described herein and may take many forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions may be constructible or additionally processable (e.g., processed into an active form), for example by adding a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. In some embodiments, the compositions may be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO and paraformaldehyde. In some embodiments, the preservation solution contains a certain amount of a metalloproteinase inhibitor.

[0090] In some embodiments, the kit contains any one or more of the components described herein in one or more containers. Therefore, in some embodiments, the kit may include a container holding the reagents described herein. The reagents may be in liquid, gel, or solid (powder) form. The reagents may be aseptically prepared, packaged in syringes, and transported frozen. Alternatively, they may be contained in vials or other containers for storage. A second container may contain other aseptically prepared reagents. Alternatively, the kit may include premixed active agents and be transported in syringes, vials, tubes, or other containers. The kit may have one or more components required to administer the reagents to a subject, such as syringes, topical application devices, or IV needles and bags.

[0091] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0092] Example

[0093] Example 1: Preparation of the 293T-B19G cell line

[0094] 1. Plasmid B19G-IRES-GFP: Based on the retroviral vector (Moloney murine leukemia virus, GenBank: AF033811.1), the target sequence is the B19G gene; plasmid gag / pol: related protein genes required for virus preparation, such as viral core proteins and enzymes required for viral replication; pCAG-VSVG: encodes envelope proteins.

[0095] 2. Cells: HEK 293T cells (Source: Cell Bank of Chinese Academy of Sciences).

[0096] 3. Experimental Procedure

[0097] 1) Preparation of B19G retrovirus:

[0098] a) Two days before transfection, add 5 ml of diluted PLL (Poly-L-lysine solution, SIGMA, catalog number: P4832, storage concentration 0.01%, dilute with 5 times the volume of DPBS to 0.01% PLL solution at a ratio of 1:5 before use) to a new 15 cm culture dish and incubate overnight at 37°C.

[0099] b) The day before transfection, remove the 15cm culture dish that has been incubated with PLL overnight and completely aspirate the PLL solution; then passage HEK-293T cells into it to ensure that the cells reach 70-80% confluency and a number of 1.5-1.8E7 / dish by the time of transfection the next day.

[0100] c) On the day of transfection, confirm that the cell density reaches 70-80% confluency, and replace the culture medium in the 15cm culture dish with 12ml. Medium (serum-depleted culture medium, Gibco, catalog number: 11058021) with DNA: L2K (Lipofectamine) TM The 2000 transfection reagent (Invitrogen, catalog number: 11668019) was used at a ratio of 1:2.3. The above plasmid without endotoxin was used to transfect 293T cells. After 5-6 hours, each culture dish was replaced with 12 ml of DMEM complete medium (Dulbecco's Modified Eagle Medium, Gibco, catalog number: C11995500BT) containing 10% fetal bovine serum.

[0101] 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 degrees Celsius for 2 hours) to obtain concentrated virus.

[0102] 2) Cell line preparation: The concentrated virus was seeded into 96-well plates with approximately 10K-20K 293T cells at MOI=100. Fluorescence was observed after 24 hours. 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.

[0103] 3) Cell line sorting: The 293T-B19G cell line was screened by flow cytometry.

[0104] The specific method for flow cytometry sorting is as follows: Cells are seeded in a 10cm culture dish; after the cells have grown into a confluent monolayer, the cell culture medium is discarded, and the cells are washed once with 3ml of DPBS (phosphate-buffered saline, Gibco, catalog number: 14190144); after discarding the DPBS, the cells are digested at room temperature for 2min with 2ml of trypsin (Gibco, catalog number: 25300054); then 4ml of DMEM containing 10% fetal bovine serum is added to terminate the digestion, the cells are resuspended, and transferred to a 15ml centrifuge tube for centrifugation at 200g for 5min at room temperature; after centrifugation, the supernatant is discarded, and 360ul of DMEM is added to resuspend the cells. 40ul of DNase I Solution (SIGMA, catalog number: D4513, storage concentration 1mg / mL, final concentration 100ug / mL) is added to remove extracellular DNA, and the cells are incubated at room temperature for 15min; after incubation, 5ml of DMEM is added to resuspend the cells, and the cells are centrifuged at 200g for 5min at room temperature; after centrifugation, 1ml of… 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 filtered and directly fed into the flow cytometry system for sorting. Positive cells were obtained after sorting and named 293T-B19G. After the cells reached a confluent monolayer, they were passaged for amplification and preservation.

[0105] 4. Results

[0106] The expression of green fluorescence was observed in the virus-infected cells, indicating that the virus was successfully prepared and could successfully infect 293T cells (see [link to relevant documentation]). Figure 1 The obtained cell lines were then sorted using flow cytometry, yielding positive cells.

[0107] 5. Conclusion

[0108] After sorting, a cell line with stable EGFP and B19G expression was obtained and named 293T-B19G (see [link to cell line]). Figure 2 ).

[0109] Example 2: Test of the ability of 293T-B19G cell line to package rabies virus

[0110] 1. Two cell lines: 293T cells and 293T-B19G cells.

[0111] 2. Six types of plasmids:

[0112] pRVΔG-4mCherry: The G protein gene sequence in the RV genome is replaced with the mCherry gene sequence.

[0113] pCAG-N: Rabies virus nucleoprotein. Phosphorylation of the N protein regulates viral transcription and replication; it binds to genomic RNA, protecting viral RNA from nuclease damage; in mature viral particles, the N protein is also a major component of the helical symmetry structure of the nucleocapsid.

[0114] pCAG-P: a rabies virus phosphoprotein that acts as a cofactor, interacting with the L protein to form a complete, active RNA polymerase, while simultaneously regulating viral RNA replication and viral packaging.

[0115] pCAG-G: Rabies virus glycoprotein, the only envelope protein of rabies virus, is responsible for binding to cell receptors, mediating viral invasion, and determining the pathogenicity and tissue tropism of the virus.

[0116] pCAG-L: The rabies virus macroprotein, the largest structural protein of the rabies virus, composed of approximately 2127 amino acid residues. It interacts with N and P proteins and, together with viral RNA, forms the nucleocapsid of the rabies virus.

[0117] pCAG-T7pol: T7 RNA polymerase, which can promote the efficient expression of target genes in the cytoplasm.

[0118] 3. Transfection reagent: Linear polyethyleneimine (PEI), storage concentration 1g / L, brand: BIOHUB, item number: 78PEI40000-1g, supplier: Shanghai Qifa Experimental Reagent Co., Ltd.

[0119] 4. Cell line packaging of RV virus

[0120] 1) Two days before transfection, add 3 ml of 1:5 diluted PLL to a new 10 cm culture dish (dilute with 5 times the volume of DPBS to 0.01% PLL solution) and incubate overnight at 37°C.

[0121] 2) The day before transfection, remove the 10cm culture dish that has been incubating PLL overnight and completely aspirate the PLL solution; then passage the cells into it to ensure that the cells reach 80-90% confluency when transfecting the next day.

[0122] 3) On the day of transfection, confirm that the cell density reaches 80-90% confluency, and replace the culture medium in the 10cm culture dish with 5ml of water. Medium, using a DNA:PEI mass ratio of 1:3, the above plasmid was transfected. The specific steps were to mix the DNA and PEI evenly in proportion and incubate for 20 minutes, then add it evenly to the cells containing culture medium. After 5-6 hours of transfection, 10 ml of DMEM containing 10% fetal bovine serum was replaced in each tray.

[0123] 4) Collect the supernatant daily for 3-10 days after transfection. 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℃.

[0124] 5. Determine the RV virus titer generated during packaging: Inoculate the supernatant into 293T cells and determine the virus titer using the FACS method.

[0125] The specific methods of FACS are as follows:

[0126] 1) One day before inoculation, seed 293T 30K / well in a 96-well plate.

[0127] 2) On the day of virus reception, dilute the cell supernatant:

[0128] "0": 33ul of virus supernatant + 297ul of DMEM, vortex to mix;

[0129] "1": 33ul of viral supernatant "0" + 297ul of DMEM, vortex to mix;

[0130] "2": 33ul of viral supernatant "1" + 297ul of DMEM, vortex to mix.

[0131] 3) 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 culture medium.

[0132] 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.

[0133] 6. Results

[0134] Eight supernatants were collected from each group on day 10 post-transfection. The titers of the daily supernatants were determined, and virus growth curves were plotted. Regarding fluorescence expression after transfection, there was no significant difference in mCherry expression of RVΔG-4mCherry on days 3, 6, and 9 post-transfection (see [link to article]). Figure 3 Based on the viral growth curves, the cell lines prepared by PEI 1:3 transfection showed significantly better virus packaging ability than the 293T cell line (see...). Figure 4 ).

[0135] 7. Conclusion:

[0136] The invented cell line demonstrates a significantly superior ability to rescue RV virus compared to 293T cells, indicating that the 293T-B19G cell line can significantly improve RV virus packaging efficiency. Repeated experiments have also proven the stability of this cell line. Initially transfected cells die during subsequent virus production, but the newly generated virus can continue to infect live B19G cells, resulting in even more virus production. Live 293T-B19G cells can continuously proliferate to produce cells containing B19G protein, allowing the rescued virus to further infect new 293T-B19G cells, releasing even more virus and achieving high-titer virus levels. The 293T-B19G cell line prepared using this invention can efficiently rescue RV virus, reducing viral passage and showing broad application prospects in the field of neural circuit tracing.

[0137] sequence of this article

[0138] SEQ ID NO:1-B19G nucleotide sequence:

[0139]

[0140] SEQ ID NO: 2 - Amino acid sequence of B19G protein:

[0141] MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYILAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYRWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPSGKCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVSMQTSNETKWCPPDKLVNLHDFRSDEIEHLVVEELVRKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEILPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVIPLVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCCRRANRPESKQRSFGGTGGNVSVTSQSGKVIPSWESYKSGGEIRL*

[0142] SEQ ID NO: 3 - Nucleotide sequence of pCAG - G:

[0143] Identical to Sequence ID: M31046.1, Range 1: 3317 to 4891

[0144]

[0145] RV genome sequence SEQ ID NO:4

[0146]

[0147] Amino acid sequence of N protein, SEQ ID NO:5

[0148] MDADKIVFKVNNQVVSLKPEIIVDQYEYKYPAIKDLKKPCITLGKAPDLNKAYKSVLSGMSAAKLNPDDVCSYLAAAMQFFEGTCPEDWTSYGIVIARKGDKITPGSLVEIKRTDVEGNWALTGGMELTRDPTVPEHASLVGLLLSLYRLSKISGQNTGNYKTNIADRIEQIFETAPFVKIVEHHTLMTTHKMCANWSTIPNFRFLAGTYDMFFSRIEHLYSAIRVGTVVTAYEDCSGLVSFTGFIKQINLTAREAILYFFHKNFEEEIRRMFEPGQETAVPHSYFIHFRSLGLSGKSPYSSNAVGHVFNLIHFVGCYMGQVRSLNATVIAACAPHEMSVLGGYLGEEFFGKGTFERRFFRDEKELQEYEAAELTKTDVALADDGTVNSDDEDYFSGETRSPEAVYTRIMMNGGRLKRSHIRRYVSVSSNHQARPNSFAEFLNKTYSSDS*

[0149] Amino acid sequence of P protein, SEQ ID NO:6

[0150] MSKIFVNPSAIRAGLADLEMAEETVDLINRNIEDNQAHLQGEPIEVDNLPEDMGRLHLDDGKSPNHGEIAKVGEGKYREDFQMDEGEDPSFLFQSYLENVGVQIVRQMRSGERFLKIWSQTVEEIISYVAVNFPNPPGKSSEDKSTQTTGRELKKETTPTPSQRESQSSKARMAAQIASGPPALEWSATNEEDDLSVEAEIAHQIAESFSKKYKFPSRSSGILLYNFEQLKMNLDDIVKEAKNVPGVTRLAHDGSKLPLRCVLGWVALANSKKFQLLVESDKLSKIMQDDLNRYTSC*

[0151] Amino acid sequence of G protein, SEQ ID NO:7

[0152] MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYILAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYRWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPSGKCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVSMQTSNETKWCPPDKLVNLHDFRSDEIEHLVVEELVRKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEILPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVIPLVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGETRL*

[0153] Amino acid sequence of the L protein SEQ ID NO:8

[0154]

[0155] Amino acid sequence of T7 RNA polymerase SEQ ID NO:9

[0156] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA*。

Claims

1. A retroviral vector comprising: (1) a nucleic acid sequence encoding B19G, wherein the amino acid sequence of B19G is set forth in SEQ ID NO: 2 or has at least 80% sequence identity thereto, or (2) a nucleic acid sequence having at least 80% sequence identity to (1) and encoding B19G set forth in SEQ ID NO: 2, or (3) a reverse complement of (1) or (2); preferably, the nucleic acid sequence of B19G is set forth in SEQ ID NO:

1.

2. A retrovirus comprising: (1) a nucleic acid sequence of B19G, wherein the amino acid sequence of B19G is set forth in SEQ ID NO: 2 or has at least 80% sequence identity thereto, or (2) a nucleic acid sequence having at least 80% sequence identity to (1) and encoding B19G set forth in SEQ ID NO: 2, and / or (3) a reverse complement of (1) or (2); preferably, the retrovirus comprises the retroviral vector of claim 1, the retrovirus further comprises a nucleic acid construct having retroviral genes required, preferably, the retroviral genes required include one or more selected from the group consisting of gag, pol, VSVG.

3. A cell which: (1) comprises, expresses and / or secretes B19G or a variant having at least 80% sequence identity thereto and retains functionality, (2) comprises a sequence of i) a nucleic acid sequence of B19G, wherein the amino acid sequence of B19G is set forth in SEQ ID NO: 2, and / or, ii) a sequence having at least 80% sequence identity to i) and encoding B19G set forth in SEQ ID NO: 2, and / or, iii) a reverse complement of i) or ii), (3) comprises a nucleic acid construct of the sequence of (2), (4) comprises the retrovirus of claim 2.

4. The cell of claim 3, wherein one or more of the following features are also included: the nucleic acid construct is a vector, preferably, the vector is a cloning vector, an integrating vector or an expression vector, more preferably, the vector is a retroviral vector; the cell is a HEK293 cell, preferably, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, HEK293SGGD cells.

5. A method of making a cell line stably expressing B19G, the method comprising: introducing into a cell a nucleic acid construct comprising a nucleic acid sequence encoding B19G, wherein the nucleic acid sequence is operatively linked to a promoter, the amino acid sequence of B19G is set forth in SEQ ID NO: 2 or has at least 80% sequence identity thereto, Preferably, the cell is a HEK293 cell, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, HEK293SGGD cells, More preferably, the cell is a 293T cell revived from culture passage 3 or more; the nucleotide sequence of the B19G is shown in SEQ ID NO:

1.

6. A cell line prepared by the method of claim 5.

7. A method of producing or rescuing a virus, comprising using the cell of claim 3 or 4 or the cell line of claim 6 to produce the virus, Preferably, the virus is a rabies virus, more preferably, the rabies virus is a G protein deleted rabies virus.

8. The method of claim 7, wherein, The method comprises: (1) introducing an RV genome into the cell or cell line containing the following proteins: rabies virus nucleoprotein, rabies virus phosphoprotein, rabies virus glycoprotein, rabies virus large protein, optionally also containing RNA polymerase, and (2) incubating the cell or cell line under conditions suitable for producing rabies virus, Preferably, the method comprises, (1) introducing an RV genome into the cell and expressing the following proteins in the cell or cell line: rabies virus nucleoprotein, rabies virus phosphoprotein, rabies virus glycoprotein, rabies virus large protein, optionally also expressing RNA polymerase in the cell line, and (2) incubating the cell or cell line under conditions suitable for producing rabies virus, More preferably, the introduction is achieved by transfection.

9. Use of the retroviral vector of claim 1, the retrovirus of claim 2, the cell of claim 3 or 4 and / or the cell line of claim 6 in the manufacture of a virus, in the improvement of virus titer, in the improvement of virus packaging efficiency, Preferably, the virus is a rabies virus.

10. A kit comprising the retroviral vector of claim 1, the retrovirus of claim 2, the cell of claim 3 or 4, or the cell line of claim 6.

11. Use of the retroviral vector of claim 1, the retrovirus of claim 2, the cell of claim 3 or 4, or the cell line of claim 6 in the manufacture of an agent for improving virus titer or improving virus packaging efficiency, Preferably, the virus is a rabies virus.

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