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Optically-controlled expression vector for insect cells and application

A technology of insect cells and expression vectors, applied in the field of bioengineering, can solve problems such as limited applications, achieve the effects of increasing the positive rate, testing quickly and stably, and improving efficiency

Active Publication Date: 2018-04-20
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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  • Optically-controlled expression vector for insect cells and application
  • Optically-controlled expression vector for insect cells and application
  • Optically-controlled expression vector for insect cells and 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 from Prof. Kevin HGardner (Motta-Mena et al., 2014)

pVP-EL222 plasmid

Gift from Prof. Kevin HGardner (Motta-Mena et al., 2014)

Gel Extraction kit

OMEGA bio-tek

Plasmid mini kit Plasmid Recovery Kit

OMEGA bio-tek

E.coli DH-5α competent

ATCC

restriction endonuclease

NEB Corporation

CIP

NEB Corporation

T4DNA ligase

NEB Corporation

Gel Imager

BioRad Corporation

[0033] List of primers used for plasmid cloning:

[0034]

[0035] 2. Experimental method

[0036] 1) Using 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 the Luc in the pC120-Luc plasmid through the EcoR1 and Xba1 restriction site...

Embodiment 2

[0048] Functional verification of insect cell light-controlled expression vector pMIB-C120-mCherry-GFP-EL222 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 adherently in a cell incubator at 28°C. When the cells grow to about 90%, directly blow the cells up gently. pass on.

[0052] 2.2 Sf9 insect cells were transfected with light-controlled expression vector in insect cells. There are two plasmids to be transfected in this experiment: pMIB-C120-mCherry-GFP-EL222 (functional plasmid) and pMIB-C120-mCherry-GFP (negative control plasmid).

[0053] 1) Press Sf9 cells 24 hours in advance by 8 × 10 5 Cells / well were seeded in 6-well plates, and transfection was carried out when the cell confluence reached 70-80%. Prepare 10ml plate medium before transfection: 1.5ml Grace cell culture medium (conta...

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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 biological engineering, and particularly 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. In the past few decades, chemically regulated gene expression systems have been widely used to temporally regulate gene expression. However, precise spatial and temporal control of gene expression is difficult due to the free diffusion of these small-molecule inducers, their intractable elimination, and their potential off-target effects on cellular function. However, light is an ideal inducer of gene expression, which is highly controllable, non-toxic, and can achieve precise temporal and spatial control of target gene expression. Thus, light-controlled gene expression systems represent a prom...

Claims

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Application Information

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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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