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Sindbis virus vector and virus particles thereof, and application of Sindbis virus vector and virus particles thereof in neural circuit

A technology of virus vectors and virus particles, applied in Sindbis virus vectors and their virus particles, and in the application field of neural circuits, can solve the problems of cumbersome steps, complicated operations, and no application of primate neural circuits, etc., to achieve Effect of saving time and operation steps

Pending Publication Date: 2021-12-21
中国科学院深圳理工大学筹
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to a series of problems in process and technical transformation, it has not yet been applied to the markers of neural circuits in rats and mice and primate neural circuits.
At present, most of the methods for obtaining Sindbis virus particles are in vitro transcription first, and then transfect cells with RNA, and use the cells to produce Sindbis virus particles. Carriers and methods

Method used

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  • Sindbis virus vector and virus particles thereof, and application of Sindbis virus vector and virus particles thereof in neural circuit
  • Sindbis virus vector and virus particles thereof, and application of Sindbis virus vector and virus particles thereof in neural circuit
  • Sindbis virus vector and virus particles thereof, and application of Sindbis virus vector and virus particles thereof in neural circuit

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Experimental program
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Effect test

Embodiment 1

[0031] The Sindbis virus vector of the present invention is prepared through the following steps:

[0032] SINV-WT (structure such as figure 1 Shown in A and 1D) and SINV-EGFP (structure such as figure 1 Shown in B and 1C) all start the transcription and translation of virus structural protein and nonstructural protein by UBC promoter, complete the packaging of virus. The primer amplification fragment shown in SEQ ID NO.2 and SEQ ID NO.3 was used to amplify the UBC promoter SEQ ID NO.4, which was inserted into the vector by SacI and PacI. Use the primers shown in SEQ ID NO.5 and SEQ ID NO.6 to amplify the EGFP gene fragment SEQ ID NO.7, the EGFP gene is initiated by the second viral subgenome promoter (26S) on the SINV vector, using SEQ ID NO. 8 and the primers shown in SEQ ID NO.9 amplified 26S, and fused with the EGFP gene in a DNA fragment (ie, SEQ ID NO.10), which was inserted into the vector via ApaI and NotI.

[0033] The PCR reaction system is 50μl: 5×Reaction Buffer...

Embodiment 2

[0037] Fluorescent expression and one-step growth curve after Sindbis virus vector transfection carrying EGFP gene of the present invention:

[0038] After extracting the SINV-EGFP plasmid of Example 1 with a plasmid extraction kit, transfect BHK-21 cells with lipofectamine 2000, 37°C, 5% (v / v) CO 2 Cultivate in an incubator to infect BHK-21 cells with the virus, and observe the cell state and fluorescence expression through an inverted fluorescence microscope at different time points. The results are as follows: figure 2 As shown in A, a green fluorescent signal can be produced after 12 hours, indicating that the Sindbis virus vector can successfully package the SINV virus and express the foreign protein efficiently and rapidly. A part of the virus supernatant collected at different time points was subpackaged, and a part was taken to detect the virus titer of the supernatant collected at different time points by double-layer plaque method, and a one-step growth curve was dr...

Embodiment 3

[0040] The Sindbis virus particles prepared in Example 2 infect various cells cultured in vitro, and can replicate and spread in the cells:

[0041] Infect the virus supernatant collected in Example 2 in BHK-21 cells and primary cultured neuronal cells respectively, take 5 μ l of the supernatant 2 days after transfection and infect the primary cultured neuronal cells and BHK-21 cells, in The expression of green fluorescence was observed 1 day (24 hours) after infection. The result is as image 3 as shown, image 3 A is the primary cultured neuron cells, image 3 B is BHK-21 cells. Embodiment 2 has proved that the Sindbis virus vector of the present invention can transfect BHK-21 cells to produce Sindbis virus particles, so image 3 The result of B can be used as a positive control. image 3 The BHK-21 cells in B are all infected by Sindbis virus and express green fluorescence, which further verifies that the Sindbis virus can infect and replicate in BHK-21 cells. image ...

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Abstract

The invention discloses a Sindbis virus vector and virus particles thereof, and application of the Sindbis virus vector and virus particles thereof in a neural circuit. The Sindbis virus particles carrying a green fluorescent protein gene are successfully prepared by transfecting cells with the Sindbis virus vector with a nucleotide sequence of SEQ ID NO. 1. The virus particles can infect a plurality of cells in vitro, can replicate and proliferate after infection, and can carry out forward cross-multistage-synapse propagation in a mouse cranial neural circuit; and the virus particles have a wide application value in the aspects of cranial neural circuit marking and tracing, marking of non-human primate nerve cells, establishment of a drug screening platform, research and development of pharmaceutical virus inhibition action mechanisms, viral vaccines and diagnostic reagents, establishment of animal models, analysis of virus replication and pathogenesis mechanisms and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Sindbis virus vector, virus particles thereof and application in neural circuits. Background technique [0002] There are many different types of neurons in the central nervous system, and their dendrites and axons together form a complex neural network. These complex neural networks enable the body to carry out normal physiological activities, such as learning, cognition, memory, fear, etc. Abnormal neural networks can also cause corresponding neurological diseases, such as Parkinson's, Alzheimer's and autism. Analyzing brain neural circuits is the basis for a deeper understanding of brain function and neurological diseases, and virus tracer tools can help analyze brain neural circuits. Therefore, the development of viral tracer tools provides additional options for mapping brain connectivity. [0003] Sindbis virus (SINV) belongs to Togaviridae and Alphavirus genus...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N7/01A61K49/00
CPCC12N15/86C12N7/00A61K49/0047A61K49/0097C12N2770/36043C12N2770/36021
Inventor 贾凡施祥玮徐富强
Owner 中国科学院深圳理工大学筹
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