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Preparation method and application of Cre and Flp dependent retrograde tracing recombinant pseudo-rabies virus

A technology of pseudorabies virus and recombinant virus, which is applied in the biological field and can solve the problems of lack of recombinant pseudorabies virus, etc.

Active Publication Date: 2018-02-16
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, there is a lack of recombinant Pseudorabies viruses that depend on Cre and Flp for expression of fluorescent proteins of different colors and cross-level transport

Method used

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  • Preparation method and application of Cre and Flp dependent retrograde tracing recombinant pseudo-rabies virus
  • Preparation method and application of Cre and Flp dependent retrograde tracing recombinant pseudo-rabies virus
  • Preparation method and application of Cre and Flp dependent retrograde tracing recombinant pseudo-rabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: a kind of Cre and Flp system-dependent reverse tracing recombinant pseudorabies virus preparation method, comprising the following steps:

[0043] 1.1 Cre and Flp-dependent clones expressing green fluorescent protein and red fluorescent protein and containing homology arms:

[0044] 1.1.1 Clones capable of Cre-dependent expression of green fluorescent protein

[0045] The PCR method was used to amplify EGFP (see SEQ ID NO.1 for the sequence) and Ubc promoter (see SEQ ID NO.2 for the sequence). The primers for amplifying the corresponding sequence are as follows respectively: primers for the DNA fragment EGFP: SEQ ID NO:3 and SEQ ID NO:4, the template is pEGFP; primers for the DNA fragment Ubc promoter: SEQ ID NO:5 and SEQ ID NO:6 , the template is FUGW(14883#, addgene). All PCR primers used in the present invention were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. First use AscI and NcoI to double cut pAAV-EF1a-double floxed-hChR2(H134R)-EY...

Embodiment 2

[0063] Example 2: A Cre and Flp system-dependent reverse tracer Recombinant pseudorabies virus expresses green fluorescent protein and red fluorescent protein depending on Cre and Flp respectively:

[0064] At present, there is no reverse visualization system that has the ability to express both Cre and Flp-dependent green fluorescent protein and red fluorescent protein in a recombinant pseudorabies virus at the same time. Fluorescent proteins and DsRed capable of analyzing:

[0065] On the one hand, by transfecting pNrl-Cre (can express Cre, 13780#, addgene) to BHK21 cells, take 5 μ l of the recombinant pseudorabies virus PRV527 prepared in Example 1 after 24 hours (the virus titer is 1.5×10 7 PFU / ml) to infect BHK21 cells expressing Cre, 37°C, 5% (v / v) CO 2 They were cultured in an incubator, and the expression of fluorescence was observed using the same exposure parameters after infection.

[0066] Experimental results:

[0067] figure 2 Middle A: Cells expressing Cre ...

Embodiment 3

[0073] Embodiment 3: A Cre and Flp system-dependent reverse tracer recombinant pseudorabies virus stability analysis:

[0074] In order to analyze the stability of PRV527 carrying Cre and Flp-dependent elements, take 5 μl of PRV527 prepared in Example 1 (the virus titer is 1.5×10 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 perform plaqueassay to detect the morphology of plaque on the one hand 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 image 3 As shown, the recombinant pseudorabies virus PRV527 was passed on BHK21 cells for 10 generations. Get the recombinant pseudorabies virus PRV527 of P10 generation to detect according to t...

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Abstract

The invention discloses a preparation method and application of a Cre and Flp dependent retrograde tracing recombinant pseudo-rabies virus. The preparation method comprises the following steps: (1) preparing a Cre and Flp system dependant retrograde tracing recombinant pseudo-rabies virus; and (2) using the virus in a labeled neural circuit, and successfully preparing a recombinant pseudo-rabies virus with independent and controllable Cre and Flp by the platform. The preparation method successfully obtains recombinant pseudo-rabies virus with independent Cre and Flp that can express fluorescent protein. The virus can be widely used in neural circuit labeling, establishment of drug screening platform, study on virus inhibiting mechanism of drugs, development of viral vaccine and diagnosticreagent, establishment of animal model, virus replication, analysis of 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 Cre and Flp system-dependent reverse tracing recombinant pseudorabies virus. 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. Therefore, it is of great significan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N7/01C12Q1/70G01N1/30G01N1/28C12R1/93
CPCC12N7/00C12N15/65C12N15/85C12N2710/16721C12N2710/16731C12N2710/16752C12Q1/025G01N1/28G01N1/30
Inventor 贾凡徐富强徐小琴缪欢吕培
Owner 布林凯斯(武汉)生物技术有限公司
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