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Combining method for optogenetics and neprilysin genes

A technology of enkephalinase and optogenetics, applied in the field of biomedical materials, can solve problems such as trauma, and achieve the effect of reducing risk and efficient degradation

Pending Publication Date: 2020-04-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods all need to implant optical fibers in the brain, which brings certain trauma to the body.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] The present invention mainly relates to the construction of plasmid, and concrete method is as follows:

[0015] 1) Construction of the enkephalinase gene and the ion channel protein gene ChR2 for light control: using the mammalian expression plasmid VR1012 as the gene carrier, the enkephalinase gene and the light-activated neuronal channel protein ChR2 gene were respectively subjected to PCR and enzyme digestion , connecting and picking single clones were recombined into the eukaryotic expression plasmid, and the nerve-specific promoter of hSyn was added in front of ChR2 to obtain the simultaneous expression of enkephalinase and light-activated neuron channel protein ChR2 in vivo and in vitro At the same time, in order to facilitate the tracing of the expression and distribution of neprilysin and ChR2, the EGFP green label was connected after the enkephalinase, and the mCherry red label was connected after ChR2. Finally, the obtained recombinant plasmid DNA was subject...

Embodiment 2

[0018] The present invention mainly relates to the construction of plasmid, and concrete method is as follows:

[0019] 1) Construction of the enkephalinase gene and the ion channel protein gene ChR2 for light control: using the mammalian expression plasmid VR1012 as the gene carrier, the enkephalinase gene and the light-activated neuronal channel protein ChR2 gene were respectively subjected to PCR and enzyme digestion , connecting and picking single clones were recombined into the eukaryotic expression plasmid, and the nerve-specific promoter of hSyn was added in front of ChR2 to obtain the simultaneous expression of enkephalinase and light-activated neuron channel protein ChR2 in vivo and in vitro At the same time, in order to facilitate the tracing of the expression and distribution of neprilysin and ChR2, the EGFP green label was connected after the enkephalinase, and the mCherry red label was connected after ChR2. Finally, the obtained recombinant plasmid DNA was subject...

Embodiment 3

[0022] The present invention mainly relates to the construction of plasmid, and concrete method is as follows:

[0023] 1) Construction of the enkephalinase gene and the ion channel protein gene ChR2 for light control: using the mammalian expression plasmid VR1012 as the gene carrier, the enkephalinase gene and the light-activated neuronal channel protein ChR2 gene were respectively subjected to PCR and enzyme digestion , connecting and picking single clones were recombined into the eukaryotic expression plasmid, and the nerve-specific promoter of hSyn was added in front of ChR2 to obtain the simultaneous expression of enkephalinase and light-activated neuron channel protein ChR2 in vivo and in vitro At the same time, in order to facilitate the tracing of the expression and distribution of neprilysin and ChR2, the EGFP green label was connected after the enkephalinase, and the mCherry red label was connected after ChR2. Finally, the obtained recombinant plasmid DNA was subject...

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Abstract

The invention discloses a combining method for optogenetics and neprilysin (NEP) genes. The method comprises the following main steps: 1) by using mammalian expression plasmid VR1012 as a gene vector,recombining the neprilysin genes and light-activated neuronal channel protein ChR2 genes to a eukaryotic expression plasmid through PCR, digestion, connection and monoclonal picking, and adding a neural specific promoter of hSyn before ChR2 to obtain a recombinant plasmid capable of simultaneously expressing neprilysin and the light-activated neuronal channel protein ChR2 in vivo and in vitro; 2)connecting an EGFP green label after the neprilysin, and connecting an mCherry red label after the ChR2; and 3) performing gene sequencing on the obtained recombinant plasmid DNA, and verifying whether the neprilysin and ChR2 genes are inserted correctly into the VR1012 eukaryotic expression plasmid at the nucleic acid molecular level. The NEP is the most important enzyme for degrading Abeta protein in a brain, so that the NEP can be used to reduce risk.

Description

technical field [0001] The invention belongs to the field of biomedical materials, and mainly designs a neprilysin (neprilysin, NEP) for gene degradation of Aβ protein and a light-sensitive protein that can open cation channels and promote cell excitation under blue light irradiation, so that optogenetics Technology combined with genetic technology. Background technique [0002] Alzheimer's disease (AD), also known as senile dementia, is a common degenerative disease of the central nervous system, the main clinical manifestations are progressive memory loss, cognitive dysfunction, personality and behavior changes and other symptoms , the specific etiology is so far unknown. Because AD is closely related to aging, with the general extension of life expectancy and the aggravation of population aging, the incidence of AD also increases, which has caused great harm to people's health and further increased medical costs. And increase the social burden. [0003] For the treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C12N15/65C12N5/10
CPCC12N9/6494C12Y304/24011C07K14/47C12N15/85C12N15/65C07K2319/00
Inventor 郑斌郭明明明东甘霖刘爽
Owner TIANJIN UNIV
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