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Preparation method and application of a highly sensitive recombinant pseudorabies virus expressing green fluorescent protein for reverse neural circuit tracing

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 good visualization.

Active Publication Date: 2019-12-24
布林凯斯(武汉)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 and application of a highly sensitive recombinant pseudorabies virus expressing green fluorescent protein for reverse neural circuit tracing
  • Preparation method and application of a highly sensitive recombinant pseudorabies virus expressing green fluorescent protein for reverse neural circuit tracing
  • Preparation method and application of a highly sensitive recombinant pseudorabies virus expressing green fluorescent protein for reverse neural circuit tracing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: A kind of preparation method of the recombinant pseudorabies virus of the reverse neural circuit tracing of expressing green fluorescent protein highly sensitively, comprises the following steps:

[0038] (1) Clones capable of highly expressing green fluorescent protein and containing homology arms:

[0039] ① Cloning with high-efficiency expression of green fluorescent protein: Firstly, 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 synthetic gene of SEQ ID NO.1 The name of the plasmid is pUC57-EGFP-F2A-EGFP-T2A-EGFP; then the PCR method is 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 method of enzyme digestion and recombination, according to figur...

Embodiment 2

[0047] Example 2: A recombinant pseudorabies virus that expresses green fluorescent protein with high sensitivity and reverse neural circuit tracer expresses green fluorescent protein efficiently:

[0048] In order to show that the present invention has obvious advantages over the existing systems in terms of the ability to express foreign proteins, this embodiment will analyze the expression level of green fluorescent protein: on the one hand, take 5 μl of the highly sensitive expression green fluorescent protein prepared in Example 1 Protein reverse neural circuit tracer recombinant pseudorabies virus PRV531 (virus titer is 1.2×10 7 PFU / ml) to infect BHK21 cells, on the other hand, take 5 μ l (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 ℃, 5% (v / v) CO 2 They were cultured in an incubator, and the expression of fluorescence was observed using the same expo...

Embodiment 3

[0050] Example 3: A highly sensitive recombinant pseudorabies virus expressing green fluorescent protein stably expressing green fluorescent protein:

[0051] In order to analyze the stability of the green fluorescent protein gene carried by PRV, get 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 the supernatant (as P1) after 2 days of infection, carry out passage on BHK21 cells for 10 generations according to the above method, collect the virus fluid of each generation, and carry out plaque assay on the one hand to detect the plaque Morphology and uniformity, on the other hand, the stability of EGFP in the process of virus passage and the ability of the virus to express fluorescence after infecting BHK21 cells in vitro were analyzed. 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 gre...

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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 specifically relates to a preparation method and application of a recombinant pseudorabies virus that expresses green fluorescent protein with high sensitivity and is traced by a reverse neural circuit. Background technique [0002] The human brain is one of the most complex systems in nature, and the neural network is the basis of the brain's functions. The normal connection of the neural network makes the human body produce normal physiological activities, such as cognition, learning, memory and fear, etc.; the abnormality of the neural network often leads 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, neither the normal physiological activities nor the pathogenic mechanism is clear, and the main reason is the lack of connection information of t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 布林凯斯(武汉)生物技术有限公司