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Preparation method and application of recombinant pseudorabies virus performing highlighted expression on reverse neural circuit tracing of red fluorescence protein

A technology of red fluorescent protein and pseudorabies virus, which is applied in the field of preparation of recombinant pseudorabies virus, can solve the problems of limited application and strong virulence, and achieve the effect of wide application value and high efficiency

Inactive Publication Date: 2019-06-28
WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the wild-type PRV is highly virulent, causing death in rats about 3 days after infection, and has the characteristics of bidirectional movement, so its application in neural circuit research is limited.

Method used

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  • Preparation method and application of recombinant pseudorabies virus performing highlighted expression on reverse neural circuit tracing of red fluorescence protein
  • Preparation method and application of recombinant pseudorabies virus performing highlighted expression on reverse neural circuit tracing of red fluorescence protein
  • Preparation method and application of recombinant pseudorabies virus performing highlighted expression on reverse neural circuit tracing of red fluorescence protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039]Embodiment 1: A kind of preparation method of the recombinant pseudorabies virus of the reverse neural circuit tracing of high brightness expression red fluorescent protein, comprises the following steps:

[0040] (1) Clones capable of highlighting the expression of red fluorescent protein and containing homology arms:

[0041] ① Cloning capable of highlighting the expression of red fluorescent protein: First, mRuby3-F2A-mRuby3-T2A-mRuby3 (see SEQ ID NO.1 for the sequence) was synthesized by whole gene synthesis, and inserted into pUC57, containing the gene of SEQ ID NO.1 The name of the synthetic plasmid is pUC57-mRuby3-F2A-mRuby3-T2A-mRuby3; 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 for the sequence). .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 cutting a...

Embodiment 2

[0049] Example 2: A recombinant pseudorabies virus stably and brightly expressing red fluorescent protein for reverse neural circuit tracing:

[0050] In order to analyze the stability of the red fluorescent protein gene carried by PRV, get 5 μl of PRV724 prepared in Example 1 (the virus titer is 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 mRuby3 during virus passage and the ability of the virus to express fluorescence after infecting BHK21 cells in vitro were analyzed. The result is as image 3 As shown, the recombinant pseudorabies virus PRV724 was passaged on BHK21 cells for 10 passages, and the results showed that it could stably express red fluorescent protein d...

Embodiment 3

[0051] Example 3: A recombinant pseudorabies virus that expresses red fluorescent protein highly efficiently expresses red fluorescent protein:

[0052] 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 red fluorescent protein: on the one hand, take 5 μl of the highly expressed red fluorescent protein prepared in Example 1 Protein reverse neural circuit tracer recombinant pseudorabies virus PRV724 (virus titer is 2.3×10 7 PFU / ml) to infect BHK21 cells, on the other hand, take 5 μ l (the virus titer of the control is 1.8×10 7 PFU / ml) pseudorabies virus PRV614 (Banfield BW et al., J Virol.2003Sep; 77(18):10106-12.) infected BHK21 cells, 37 ℃, 5% (v / v) CO 2 After culturing in an incubator, the same exposure parameters were used to observe the expression of fluorescence after infection. The results showed that the brightness of the f...

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Abstract

The invention discloses a preparation method and application of a recombinant pseudorabies virus performing highlighted expression on reverse neural circuit tracing of red fluorescence protein. The method comprises the steps of 1 preparation of the recombinant pseudorabies virus expressing the red fluorescent protein and 2 application in labeling a neural circuit, and the recombinant pseudorabiesvirus performing highlighted expression on the red fluorescent protein is successfully obtained through a platform. Accordingly, the recombinant pseudorabies virus performing highlighted expression onthe reverse neural circuit tracing of the red fluorescence protein is successfully obtained, and the method and application have wide application value in the aspects of neural circuit labeling, establishment of a drug screening platform, action mechanism of inhibiting the virus through drugs, development of a viral vaccine and a diagnostic reagent, establishment of an animal model, virus replication, analysis of a pathogenic mechanism 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 highlights red fluorescent protein and traces 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 the brain neural network. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85A61K49/00A61K35/76C12R1/93
Inventor 贾凡徐富强李莉缪欢吕培施祥玮
Owner WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI