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Preparation method and application of attenuated rabies virus

A rabies virus and virus technology, applied in the biological field, can solve the problems of strengthening neurons, low titer, difficult preparation, etc., achieve wide application value and market prospects, and improve the effect of trans-synaptic efficiency

Active Publication Date: 2021-02-02
INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reardon et al. established a retrograde transsynaptic system modified by the CVS-N2c strain of rabies virus. After the genome has lost its own glycoprotein N2cG, by reverse compensating N2cG, its retrograde transsynaptic ability is obvious compared with the SAD-B19 vaccine strain Enhanced and further reduce the toxicity to neurons, and can express functional probes such as calcium ion probes and optogenetic probes for the analysis of functional circuits, so it has more advantages in neural circuit tracing, but there are also very difficult preparations. Difficulty, low titer and other shortcomings, so there are no related reports on its use in loop tracing, and it is unknown whether oG can combine with CVS-N2c-ΔG to improve transsynaptic efficiency

Method used

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  • Preparation method and application of attenuated rabies virus
  • Preparation method and application of attenuated rabies virus
  • Preparation method and application of attenuated rabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Preparation method of attenuated rabies virus CVS-N2c-ΔG

[0018] 1. Establishment of BHK-EnvARVG stable cell line

[0019] Using the DIO-EF1a-FLEX-H2B-GFP-P2A-N2cG plasmid (addgene #73476) as a template, the H2B-GFP-P2A fragment was amplified with primers H2B-GFP-P2A-F and H2B-GFP-P2A-R , its nucleotide sequence is shown in SEQ ID NO.1; with the PMD-19T-EnvARVG plasmid (provided by Wuhan Privy Brain Science and Technology Co., Ltd.) as a template, it is amplified with primers 2A-EnvARVG-F and 2A-EnvARVG-R Obtain the 2A-EnvARVG fragment, the nucleotide sequence of which is shown in SEQ ID NO.2; the first two fragments obtained are used as templates, and the fusion PCR amplification is carried out with primers H2B-GFP-P2A-F and 2A-EnvARVG-R Obtain the H2B-GFP-P2A-EnvARVG fragment, the nucleotide sequence of which is shown in SEQ ID NO.3; use the kit One Step Cloning Kit (Vazyme Company) for recombinant cloning to connect the H2B-GFP-P2A-EnvARVG into BamHI and Ec...

Embodiment 2

[0052] Example 2 Comparison of cross-synaptic efficiency of CVS-N2c-ΔG-tdTomato and SAD-B19-ΔG-DsRed in combination with oG

[0053] Combining different origins of virus strains with different origins of outer membrane glycoproteins may have different transsynaptic efficiencies. Using codon-optimized chimeric glycoprotein (oG) derived from rabies virus Pasteur strain can assist SAD-B19-ΔG-DsRed to greatly improve the trans-synaptic tracing efficiency, and then SAD-B19-ΔG-DsRed / oG crosses single synapses Synaptic systems are widely used to track the upstream input of neural networks. However, whether oG can assist CVS-N2c-ΔG to cross synapses and how efficiently has not been reported. Therefore, it is necessary to compare different retrograde transmonosynaptic systems, combining AAV-Ef1a-DIO-H2B-GFP-2A-oG-WPRE-hGH and AAV-Ef1a-DIO-TVA- The helper virus of WPRE-hGH (provided by Wuhan Privy Brain Science and Technology Co., Ltd.) was mixed and injected into the ventral hippocam...

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Abstract

The invention discloses a preparation method and application of attenuated rabies virus. The preparation method comprises the following steps of firstly, constructing a cell line BHK-EnvARVG for stably expressing nuclear localization green fluorescence and EnvARVG protein, quickly rescuing and preparing CVS-N2c-[delta]G-tdTomato virus by using a cell line B7GG, and then infecting the cell line BHK-EnvARVG to prepare high-titer EnvARVG pseudotype CVS-N2c-[delta]G-tdTomato virus. Compared with the prior art, the method solves the problem that CVS-N2c-[delta]G virus is difficult to produce, the transsynaptic efficiency can be further improved through a CVS-N2c-[delta]G-tdTomato / oG system, and the transsynaptic efficiency of the CVS-N2c-[delta]G-tdTomato / oG system is 3-4 times that of an SAD-B19-[delta]G-DsRed / oG system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of an attenuated rabies virus. Background technique [0002] Anterograde and retrograde viral tracers are required for neural circuit tracing and functional studies. Using reverse genetics technology, Wickersham et al. obtained recombinant rabies virus infectious clones based on the RV vaccine strain SAD-B19, deleted the RVG gene encoding the envelope glycoprotein, and inserted multiple cloning sites, which can carry various fluorescent genes, etc. The source gene is expressed in high abundance in neurons, thereby clearly marking the fine morphology of neurons. Using the principle that the envelope glycoprotein (EnvA) of avian sarcoma virus (ASLV) specifically recognizes its receptor protein TVA, the extramembrane region of EnvA is fused with the transmembrane region and the intracellular region of RVG to construct an expression inser...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/867
CPCC12N7/00C12N15/86C12N2760/20121C12N2740/15043
Inventor 林坤章徐富强
Owner INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
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