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Preparation method of recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity, and application

A technology of green fluorescent protein and pseudorabies virus, applied in the biological field, can solve the problem of low efficiency of fluorescent protein, and achieve the effect of wide application value, visualization and high efficiency

Active Publication Date: 2017-08-01
衠奥生物技术(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Nevertheless, the efficiency of expressing fluorescent protein is low, therefore, it is of great significance to establish a recombinant pseudorabies virus expressing green fluorescent protein with high sensitivity

Method used

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  • Preparation method of recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity, and application
  • Preparation method of recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity, and application
  • Preparation method of recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity, and application

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1: A preparation method of recombinant pseudorabies virus traced by reverse neural circuit tracer expressing green fluorescent protein with high sensitivity, comprising the following steps:

[0038] (1) Clones with high-efficiency expression of green fluorescent protein and containing homology arms:

[0039] ①Clone capable of highly expressing green fluorescent protein: First, EGFP-F2A-EGFP-T2A-EGFP (see SEQ ID NO.1 for the sequence) was synthesized by whole gene synthesis, and inserted into pUC57, containing the synthesis of SEQ ID NO.1 The plasmid name is pUC57-EGFP-F2A-EGFP-T2A-EGFP; then PCR was used to amplify the CAG promoter (see SEQ ID NO. 2 for the sequence) and the rabbit β-globin intron (see SEQ ID NO. 3), WPRE (see SEQ ID NO. 4 for the sequence) and BGHpA (see SEQ ID NO. 5 for the sequence), so as to obtain the respective PCR fragments, and adopt the mode of enzyme cleavage and recombination, according to figure 1 The CAG promoter, rabbit β-globi...

Embodiment 2

[0047] Example 2: A highly sensitive recombinant pseudorabies virus expressing green fluorescent protein with reverse neural circuit tracer High expression of green fluorescent protein:

[0048] In order to show that the present invention has obvious advantages over the existing system in terms of the ability to express exogenous proteins, this example will analyze the expression level of green fluorescent protein: on the one hand, take 5 μl of the highly sensitive expression green fluorescent Reverse neural circuit tracing of protein recombinant pseudorabies virus PRV531 (virus titer was 1.2 × 10 7 PFU / ml) to infect BHK21 cells, on the other hand, take 5 μl (the virus titer of the control is 1.5×10 7 PFU / ml) pseudorabies virus PRV152 (Smith BN et al., Proc Natl Acad Sci US A. 2000, 97(16):9264-9.) infected BHK21 cells, 37°C, 5% (v / v) CO 2 The cells were cultured in an incubator, and the fluorescence expression was observed using the same exposure parameters after infection. ...

Embodiment 3

[0050] Example 3: A highly sensitive recombinant pseudorabies virus expressing green fluorescent protein with reverse neural circuit tracing stably expressing green fluorescent protein:

[0051] In order to analyze the stability of PRV carrying the green fluorescent protein gene, take 5 μl of PRV531 prepared in Example 1 (the virus titer is 1.2×10 7 PFU / ml) (as P0) to infect BHK21 cells, collect supernatant (as P1) after 2 days of infection, pass on BHK21 cells for 10 generations according to the above method, collect the virus liquid of each generation, on the one hand, carry out plaque assay to detect plaque Morphology and homogeneity, on the other hand, analyzed the stability of EGFP during virus passage and the ability of the virus to express fluorescence after infecting BHK21 cells in vitro. The result is as Figure 4 As shown, the recombinant pseudorabies virus PRV531 was passaged on BHK21 cells for 10 passages, and the results showed that it could stably express green ...

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Abstract

The invention discloses a preparation method of a recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity, and application, including (1) the preparation of the recombinant pseudorabies virus for expressing green fluorescin with high sensitivity; (2) the application to marking of a neural circuit. The recombinant pseudorabies virus for expressing the green fluorescin with high sensitivity is successfully prepared by using a platform. The recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity is successfully obtained. Wide application value is realized in aspects of neural circuit marking, medicine screening platform building, medicine inhibition virus action mechanism, viral vaccine and diagnostic reagent invention and development, animal model building, virus replication, pathogenic mechanism analysis and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more particularly relates to a preparation method and application of a recombinant pseudorabies virus traced by a reverse neural circuit tracer expressing green fluorescent protein with high sensitivity. Background technique [0002] The human brain is one of the most complex systems in nature, and the neural network is the basis for the functioning of the brain. The normal connection of the neural network enables the human body to produce normal physiological activities, such as cognition, learning, memory and fear; abnormal neural networks often lead to the emergence of neurological diseases, such as: Alzheimer's disease, Parkinson's disease, depression etc., but there is no effective means to treat these neurological diseases. At present, the normal physiological activities and pathogenic mechanisms are not clear, the main reason is the lack of connection information of the brain neural netwo...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N7/01C12Q1/70G01N21/64G01N1/28G01N1/36G01N1/42C12R1/93
CPCC12N7/00C12N15/65C12N15/85C12N2710/16721C12N2710/16751C12N2800/107C12Q1/025G01N1/286G01N1/36G01N1/42G01N21/6458G01N2001/2873
Inventor 贾凡徐富强徐小琴缪欢
Owner 衠奥生物技术(武汉)有限公司
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