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A light-controlled expression vector for insect cells and its application

A technology of insect cells and expression vectors, which can be used in the field of bioengineering and can solve problems such as limited applications

Active Publication Date: 2020-10-27
布林凯斯(深圳)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

Further, scientists tried to verify the feasibility of the light-controlled system in zebrafish. Although light-controlled expression can be achieved, the application of this system in zebrafish is limited due to certain toxicity.

Method used

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  • A light-controlled expression vector for insect cells and its application
  • A light-controlled expression vector for insect cells and its application
  • A light-controlled expression vector for insect cells and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Construction of Insect Cell Expression Vector pMIB-C120-mCherry-GFP-EL222

[0031] 1. Experimental materials

[0032] pMIB / v5-hisA vector Invitrogen pC120-Luc plasmid Gift of Professor Kevin HGardner (Motta-Mena et al., 2014) pVP-EL222 plasmid Gift of Professor Kevin HGardner (Motta-Mena et al., 2014) Gel Extraction kit gel recovery kit OMEGA bio-tek company Plasmid mini kit plasmid recovery kit OMEGA bio-tek company E.coli DH-5α competent ATCC restriction endonuclease NEB Corporation CIP NEB Corporation T4DNA ligase NEB Corporation gel imager BioRad

[0033] List of primers used for plasmid cloning:

[0034]

[0035] 2. Experimental method

[0036] 1) Use the pLV-mCherry (purchased from Addgene) vector as a template, use primers E-mch-F and X-mch-R to amplify the mcherry gene fragment, and replace Luc in the pC120-Luc plasmid with EcoR1 and Xba1 restriction sites Gene, get pC120-mcherry...

Embodiment 2

[0048] Functional verification of light-controlled expression vector pMIB-C120-mCherry-GFP-EL222 in insect cells in Sf9 insect cells: 1. Experimental materials

[0049]

[0050] 2. Experimental methods and results

[0051] 2.1 Subculture of Sf9 cells: Use Grace's Insect Cell Medium plus serum and double antibody medium to culture Sf9 cells on the wall at 28°C in a cell incubator. When the cells grow to about 90%, directly blow the cells up gently pass on.

[0052] 2.2 Insect cells Light-controlled expression vectors were transfected into Sf9 insect cells. There are two plasmids that need to be transfected in this experiment: pMIB-C120-mCherry-GFP-EL222 (functional plasmid) and pMIB-C120-mCherry-GFP (negative control plasmid).

[0053] 1) Sf9 cells were divided into 8×10 cells 24 hours in advance 5 Cells / well were seeded in a 6-well plate, and transfection was performed when the confluence of the cells reached 70-80%. Prepare 10ml plate medium before transfection: 1.5ml ...

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Abstract

The invention discloses an optically-controlled expression vector for insect cells and application. An applicant successfully constructs a positive plasmid pMIB-C120-mCherry-GFP-EL222 and a control plasmid pMIB-C120-mCherry-GFP on an insect cell expression vector pMIB / V5-HisA based on a VP-EL222 optically-controlled transcription activating system; after the positive plasmid pMIB-C120-mCherry-GFP-EL222 is transfected with Sf9 cells, a green fluorescent protein expresses while a red fluorescent protein does not express; after the positive plasmid pMIB-C120-mCherry-GFP-EL222 is irradiated and stimulated by blue light, the red fluorescent protein expresses; after the control plasmid pMIB-C120-mCherry-GF is transfected with the Sf9 cells, the green fluorescent protein expresses while the red fluorescent protein does not express; after the control plasmid pMIB-C120-mCherry-GF is irradiated and stimulated by the blue light, the red fluorescent protein still does not express due to no EL222 transcription factor. An optically-controlled inducible expression system is successfully applied to the insect cells for the first time, and the application range of the technology is widened. According to a verification method constructed by the invention, the plasmids also provide a flexible and easy-to-use method for using an optically-controlled induced expression technology.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a light-controlled expression vector of insect cells and its application. Background technique [0002] With the development of transgenic technology, controllable gene expression regulation system has become an indispensable tool in biomedical research and biotechnology. Over the past few decades, chemically regulated gene expression systems have been widely used to temporally regulate gene expression. However, spatially and temporally precise control of gene expression is difficult due to the free diffusion of these small molecule inducers, difficulty in elimination and potential off-target effects on cellular function. However, light is an ideal gene expression inducer, which is highly controllable, non-toxic, and can achieve double precise control of the expression of the target gene in time and space. Therefore, light-controlled gene expression systems represent a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/65
CPCC12N15/65C12N15/8509C12N2015/8518
Inventor 吴阳徐富强何晓斌蔡泽蕲蒋良玉梅婷尚敏
Owner 布林凯斯(深圳)生物技术有限公司
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