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CN-122012397-A - Cell line for efficiently propagating EnvA pseudorabies viruses as well as preparation method and application thereof

CN122012397ACN 122012397 ACN122012397 ACN 122012397ACN-122012397-A

Abstract

The invention provides a cell line for efficiently propagating EnvA pseudorabies viruses, and a preparation method and application thereof. In particular, the invention provides a BHK cell line having an exogenous expression cassette for expressing EnvA and a fluorescent reporter gene integrated into the genome of the cell, and a method of making the cell line. The cell line of the invention can be used for packaging EnvA pseudorabies viruses, and the produced EnvA pseudorabies viruses can be used for marking and tracking specific types of neurons and researching nerve pathways.

Inventors

  • WANG XIAO
  • GAO XUEPING
  • ZHANG XIA
  • JIN LEI

Assignees

  • 临港国家实验室

Dates

Publication Date
20260512
Application Date
20241111

Claims (10)

  1. 1. A cell line for the efficient proliferation of EnvA pseudorabies virus, characterized in that the cell line is a BHK cell line and that the cell genome of the cell line has integrated therein an exogenous expression cassette expressing EnvA and a fluorescent reporter gene.
  2. 2. The cell line of claim 1, wherein the cell line for efficient proliferation of EnvA pseudorabies virus is a polyclonal cell line.
  3. 3. The cell line of claim 1, wherein the fluorescent reporter gene comprises EGFP, GFP, BFP, mCherry, tdTomato, or a combination thereof.
  4. 4. The cell line of claim 1, wherein the exogenous expression cassette has a structure according to formula I: Z1-Z2-Z3-Z4-Z5(I) In the formula, Z1 is a promoter or a promoter-containing 5' -UTR element; z2 is absent or optional enhancers; z3 is the nucleotide sequence of EnvA genes; Z4 is the nucleotide sequence of the fluorescent reporter gene, and Z5 is the none or 3' -UTR element.
  5. 5. The cell line of claim 1, wherein the cell line expresses moderate fluorescence, wherein the "moderate fluorescence" means that the fluorescence intensity is the second highest for the entire sorted cell population and the proportion of such cells is 4-5% of the entire sorted cell population.
  6. 6. A method of preparing a cell line for the efficient proliferation of EnvA pseudorabies virus according to any one of claims 1-5, characterized in that the method comprises the steps of: (a) Preparing a viral vector containing an exogenous expression cassette expressing EnvA and a fluorescent reporter gene; (b) Infecting BHK cells with the viral vector obtained in step (a) to obtain BHK cells after infection, and (C) And (3) carrying out passage amplification on the infected BHK cells in the step (b), and then screening by adopting a flow sorting method to obtain a BHK cell population expressing medium fluorescence intensity, namely the cell line for efficiently propagating EnvA pseudorabies viruses.
  7. 7. The method of claim 6, wherein in step (c), the flow sorting comprises sorting the fluorescence-positive BHK cells detected by the machine into two cell clusters, respectively a high-intensity fluorescent group and a medium-intensity fluorescent group, wherein the high-intensity fluorescent group is the highest fluorescence intensity of 1% -2% (preferably, 1% -1.5%, more preferably, 1.2% -1.5%) of the whole sorted cell population, the medium-intensity fluorescent group is the second highest fluorescence intensity of 4% -6% (preferably, 4% -5%, more preferably, 4.5% -5%) of the whole sorted cell population, and finally sorting the medium-intensity fluorescent group cell population, i.e., the cell line of interest.
  8. 8. A method of packaging EnvA pseudorabies virus, comprising packaging using the cell line of any one of claims 1-5.
  9. 9. The method according to claim 8, wherein the method comprises the steps of: (S1) infection of a cell line according to any one of claims 1 to 5 with G-protein-deleted rabies virus (RV- ΔG), and (S2) culturing the infected cell line, thereby obtaining EnvA pseudorabies virus.
  10. 10. Use of the cell line of any one of claims 1-5 for packaging EnvA pseudorabies viruses, thereby for the preparation of reagents for reverse "trans-unipolar synaptic" tracking of neurons.

Description

Cell line for efficiently propagating EnvA pseudorabies viruses as well as preparation method and application thereof Technical Field The invention relates to the technical field of biology, in particular to a cell line for efficiently propagating EnvA pseudorabies viruses, a preparation method and application thereof. Background Rabies virus (Rabies virus, RV) is one of the usual tracer tools for neuroscientists. Wild rabies virus belongs to the genus rabies virus (Lyssavirus) of the family Rhabdoviridae (Rhabdoviridae), and has an envelope on the surface, and the genome is single-stranded negative-strand RNA. The viral genome is about 12kb in length, and from 3 'to 5' there are a total of N, P, M, G genes, encoding nucleoprotein, phosphoprotein, matrix protein, glycoprotein and transcriptase macroprotein, arranged sequentially. By reverse genetics, the glycoprotein gene G responsible for viral entry was deleted from the genome and designated as first-generation rabies virus (Δg). The envelope Glycoprotein (GP) of RV is a protein necessary for retrograde and trans-synaptic, a large amount of its receptor is distributed at the tail end of axon, and after infection, the protein can retrograde along the axon into the cytoplast of neuron to start virus replication, and the RV (RV-delta G) with G protein deletion can lose the trans-synaptic capacity, and the replication and transcription are not affected (the sustainable high abundance expression of exogenous genes). The first generation rabies virus has two application scenes, namely, firstly, the original glycoprotein B19G-coated delta G virus can reversely label neurons to realize the function of reverse labeling, and secondly, the reverse 'cross-single-stage synapse' tracking of specific initial cells is realized by exogenously expressing B19G glycoprotein and TVA (a specific cell surface receptor of poultry and no endogenous expression in a mammal nervous system) in the initial cells of interest, and simultaneously, envA (outer membrane protein of avian sarcoma virus and specific recognition receptor TVA) is used for coating the rabies virus. Since mammalian cells do not have the EnvA receptor TVA, envA-coated recombinant deletion rabies virus cannot directly infect mammalian cells. Only when exogenous TVA is expressed on specific nerve cells can the EnvA-coated rabies virus vector specifically infect the nerve cells, and after replication and transcription, the vector crosses synapses under the coating of B19G to infect the upper-stage neurons, while the upstream neurons do not express B19G, and the viruses jump only stay on the neurons and cannot jump to the second-stage neurons at the upstream, which is called as the tracking of 'crossing synapses'. The Cre transgenic mouse is combined with Cre-LoxP to control the AAV helper virus for expressing TVA and G proteins, so that the TVA and G proteins can be expressed only in specific neurons in specific regions, and reverse cross-single-stage synaptic marking of specific neurons can be realized by RV-delta G (EnvA). This approach is widely used in the study of neural pathways, but the biggest challenge of this technology is the need to multiply EnvA pseudorabies viruses in large quantities, resulting in high titers of virus for animal experiments. However, similar cell lines reported to date have low toxigenic titers, and there is a need in the art for cell lines that can more efficiently proliferate EnvA pseudorabies viruses. Disclosure of Invention The invention aims at providing a cell line capable of efficiently propagating EnvA pseudorabies viruses, a method for preparing the cell line and application of the cell line. In a first aspect of the invention, there is provided a cell line for the efficient proliferation of EnvA pseudorabies virus, which is a BHK cell line, and which has integrated into its cell genome an exogenous expression cassette for the expression of EnvA and a fluorescent reporter gene. In another preferred embodiment, the cell line for efficient proliferation of EnvA pseudorabies virus is a polyclonal cell line. In another preferred embodiment, the nucleotide sequence of EnvA gene is shown in SEQ ID NO. 1. In another preferred example, the EnvA gene encodes EnvA protein with the amino acid sequence shown in SEQ ID NO. 2. In another preferred embodiment, the fluorescent reporter gene comprises EGFP, GFP, BFP, mCherry, tdTomato, or a combination thereof. In another preferred embodiment, the fluorescent reporter is EGFP. In another preferred embodiment, the nucleotide sequence of the fluorescent reporter EGFP is shown in SEQ ID NO. 3. In another preferred example, the amino acid sequence of EGFP encoded by the fluorescent reporter EGFP is shown as SEQ ID NO. 4. In another preferred embodiment, the exogenous expression cassette has a structure according to formula I: Z1-Z2-Z3-Z4-Z5 (I) In the formula, Z1 is a promoter or a promoter-containing 5' -UTR element; z2 is absent or optional enha