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A simenlik forest virus replicon and its use for sparse or fine neuronal labeling

A Ximenlik and replicator technology, applied in the biological field, can solve the problems of primate neural circuits without the application of fine cell morphology

Active Publication Date: 2020-07-21
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, due to a series of problems in process and technological transformation, it has not been applied to the depiction of fine cell morphology and the labeling of primate neural circuits.

Method used

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  • A simenlik forest virus replicon and its use for sparse or fine neuronal labeling
  • A simenlik forest virus replicon and its use for sparse or fine neuronal labeling
  • A simenlik forest virus replicon and its use for sparse or fine neuronal labeling

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Experimental program
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Effect test

Embodiment 1

[0050] A kind of Ximenlik forest virus replicon is prepared by the following steps:

[0051] 1. Using pEGFP-N2 (Invitrogen Company) as a template to amplify the EGFP gene, its sequence is SEQ ID NO: 1, using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 to amplify the fragment SEQ ID NO: 1; pSFV-replicon (Lundstrom, K et al., (2003) Mol.Ther.7, 202-209.) is used as a template to amplify the N-terminal 102bp of capsid (ie C34), its sequence is SEQ ID NO: 4, using SEQ ID NO : 5 and the primer amplified fragment SEQ ID NO: 4 shown in SEQ ID NO: 6; detect and recover the amplified DNA fragment in a conventional manner. C34 and EGFP are connected by F2A, its sequence is SEQ ID NO: 7, and its sequence is contained in primers SEQ ID NO: 6 and SEQ ID NO: 2.

[0052] The PCR reaction system is 50μl: 5×Reaction Buffer: 10μl, 10mM dNTPs: 1μl, 10μM Forward Primer: 2.5μl, 10μM Reverse Primer: 2.5μl, Template DNA: 0.5μl, DNAPolymerase: 0.5μl, Nuclease-Free Water: 33μl . The amplific...

Embodiment 2

[0055] Application of a Simeliek Forest virus replicon in the preparation of a single infectious Simmenlik Forest virus virus-like particle expressing green fluorescent protein:

[0056] NruI was used to digest pSFV-replican-EGFP and pSFV-helper (Berglund, P et al., (1993) Biotechnology 11, 916-920.), and the digested pSFV-replican-EGFP and pSFV were transcribed in vitro using MEGAscript SP6Transcription Kit -helper is RNA, and transfected into BHK21 cells, 37°C, 5% (v / v) CO 2 Cultured in an incubator, the cell state and fluorescence expression were observed by an inverted fluorescence microscope, and the virus supernatant was collected at different times after infection.

[0057] figure 2 Middle A: BHK21 cells without RNA transfection, used as the control of the experiment;

[0058] figure 2 Middle B: BHK21 cells co-transfected with pSFV-replican-EGFP and pSFV-helper RNA can produce a green fluorescent signal after 1 day, indicating that the foreign protein can be expres...

Embodiment 3

[0062] Semenlik Forest virus virus-like particles produced by the Simenlik Forest virus replicon are single-infectious:

[0063] The characteristics of a single infection of activated and inactivated Simenlik forest virus virus-like particles were analyzed separately. On the one hand, 10 μl of inactivated virus-like particles were taken to infect BHK21 cells, and the expression of green fluorescence was observed 1 day after infection, and collected Medium supernatant. Take 100 μl of the collected medium supernatant to infect BHK21 cells, observe the expression of fluorescence at different times after infection; on the other hand, take 1 μl of virus-like particles to infect BHK21 cells, observe the expression of green fluorescence 1 day after infection, and collect and culture base supernatant. 100 μl of the collected medium supernatant was taken to infect BHK21 cells, and the expression of fluorescence was observed at different times after infection.

[0064] image 3 Middl...

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Abstract

The invention discloses a semliki forest virus replicon and application of semliki forest virus replicon in sparse or fine neuron labeling. An SFV (Semliki Forest Virus) which carries a green fluorescent protein gene and has single infectivity is successfully prepared by using the replicon as shown in SEQ ID NO.8; the host infection efficiency of the virus can be controlled, so that fine labeling for mouse brain nerve cells can be realized. The emliki forest virus replicon successfully obtains the SFV which carries the green fluorescent protein gene and has the single infectivity, and has a wide application value in the aspects of description of fine forms of the cells, neural circuit labeling, labeling of inhuman primate nerve cells, construction of a drug screening platform, research and development of a drug inhibition virus action mechanism, a viral vaccine and a diagnosis reagent, building of an animal model, analysis of virus replication and a pathogenic mechanism and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a replicon of Semenlik Forest virus and its application in sparse or fine neuron labeling. Background technique [0002] The human brain is one of the most complex systems in nature, and the neural network is the basis for the brain to perform functions. The normal connection of the neural network enables the body to produce normal physiological activities, such as cognition, learning, memory and fear; the neural network Abnormalities often lead to the emergence of neurological diseases for which there is no effective means to treat them. 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 significance to carry out the study of brain neural circuits and draw high-precision brain functional connectivity maps for un...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N7/04A61K49/00C12R1/93
Inventor 贾凡徐富强朱续涛靳森曾绍群刚亚栋
Owner WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI