Rabies virus labeling whole brain area neural network structure, and preparation and application thereof

A technology of rabies virus and virus, which is applied in the field of stable cell line construction, can solve the problem of large inflammatory response at the injection site, and achieve the effects of high virus production yield, reduced inflammatory response, and high reverse projection efficiency

Active Publication Date: 2018-08-03
BRAINVTA (WUHAN) CO LTD
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  • Claims
  • Application Information

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Problems solved by technology

[0005] The existing technology lacks the RV virus system that can clearly label the fine morphology of neurons and efficiently reverse label ot

Method used

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  • Rabies virus labeling whole brain area neural network structure, and preparation and application thereof
  • Rabies virus labeling whole brain area neural network structure, and preparation and application thereof
  • Rabies virus labeling whole brain area neural network structure, and preparation and application thereof

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preparation example Construction

[0047] Correspondingly, the present invention provides a recombinant defective rabies virus for labeling nerves in brain regions, which is an attenuated strain of SAD-B19, and its viral capsid contains envelope glycoprotein N2C (G), and the envelope glycoprotein N2C(G) is the envelope glycoprotein of virus strain CVS. The preparation method of the recombinant defective rabies virus comprises the steps of:

[0048] (1) When BHK-N2C(G) cells are stable in passage, digest the cells with 0.05% or 0.25% trypsin for 2 to 3 minutes, pass the cells according to the ratio of 1:5 to 1:7, and carry out in an incubator at 35 to 37°C nourish;

[0049] (2) Cultivate for 4-6 hours, discard the medium in the culture bottle, add 13-15 mL of frozen-thawed and filtered RV-△G-dsred virus to infect, and transfer to a 33-35°C incubator to continue culturing;

[0050] (3) 48-72 hours after infection, when the infection ratio reaches more than 80%, the virus cells are digested and amplified, and cu...

Embodiment 1

[0054] (1) Construction of BHK-N2C(G) stable cell line

[0055] (1-1) FUGW-H2B-GFP-P2A-N2C(G) Lentiviral Vector Construction and Viral Packaging

[0056] FUGW was double-cut with BamHI and EcoRI to obtain FUGW (B / E), with 73476 (addgene number) as the template, H2B-GFP-P2A-N2C (G)-F (same) and H2B-GFP-P2A-N2C (G )-R(same) is the primer, the annealing temperature is 58 degrees, and the H2B-GFP-P2A-N2C(G) fragment is amplified in 3 minutes, its gene sequence is shown in SEQ ID NO: 1, FUGW(B / E) and The H2B-GFP-P2A-N2C(G) fragment was homologously recombined and cultured in a 37°C incubator. After overnight, colony PCR identification was performed, and the positive clones were subjected to plasmid extraction, enzyme digestion verification and sequencing to obtain the target plasmid FUGW-H2B- GFP-P2A-N2C(G); wherein, H2B is histone H2B with nuclear localization function, its gene sequence is shown in SEQ ID NO: 2, GFP is green fluorescent protein, its gene sequence is shown in SEQ...

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Abstract

The invention belongs to the field of construction of stable cell lines, and more specifically, relates to a rabies virus labeling a whole brain area neural network structure, and preparation and an application thereof. A third-generation lentivirus technology is used for completing preparation of a BHK-N2C(G) cell line, that is to say, through construction of an FUGW-H2B-GFP-P2A-N2C(G) lentivirusvector and virus packaging, BHK cells are infected to obtain BHK-N2C(G) cells with nuclear localization fluorescence, and the cells are used for production of the defective recombinant rabies virus.The BHK-N2C(G) cells are infected with an RV-[delta]G-X virus solution, and then an RV-N2C(G)-[delta]G-X product is prepared by amplification and purification. In-vivo test results indicate that the prepared recombinant defective rabies virus RV-N2C(G)-[delta]G-X has no obvious inflammation reaction at an injection site, and has more reverse projection areas and high reverse projection efficiency.

Description

technical field [0001] The invention belongs to the field of stable cell line construction, and more specifically relates to a rabies virus that marks the neural network structure of the whole brain region, its preparation and application. Background technique [0002] The brain neural network is a complex structure composed of a large number of neurons with different shapes and characteristics connected through synapses. It is the structural basis for the brain to perform cognition, emotion, memory, imagination and other activities. Unraveling the structure of the brain's neural network is the basic premise for ultimately understanding the mechanism of the brain's information processing. Traditional neural network tracing methods, such as electron microscopy, Golgi staining, dyes, protein and peptide markers, can show the morphology of neurons in one brain area and their projections to other brain areas, and the use of transgenic mice can also show the neurons in the brain....

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0686C12N15/86C12N2510/00C12N2740/15043
Inventor 黄晓苹杨甜高娟刘宏均
Owner BRAINVTA (WUHAN) CO LTD
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