Xenopus laevis oocyte expression carrier with yellow or red fluorescence protein label and application

A red fluorescent protein and expression vector technology, applied in the fields of molecular biology and genetic engineering, can solve the problem of consuming a lot of time and economic costs, observing whether the expression of the target protein is dynamic in time and space, and unable to purify and identify the target protein in subcellular localization. and other problems to achieve the effect of facilitating extraction and purification and simplifying the experimental flow

Active Publication Date: 2018-12-21
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Using the pGH19 plasmid to express the protein in Xenopus laevis oocytes can detect its physiological and pharmacological properties through electrophysiological experiments, but it is impossible to observe the expression of the target protein under a microscope and its dynamics in time and space, and it is impossible to perform effective subcellular localization and the purification and identification of the target protein, and the acquisition of specific antibodies against the target protein also consumes a lot of time and economic costs

Method used

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  • Xenopus laevis oocyte expression carrier with yellow or red fluorescence protein label and application
  • Xenopus laevis oocyte expression carrier with yellow or red fluorescence protein label and application
  • Xenopus laevis oocyte expression carrier with yellow or red fluorescence protein label and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of pGH19-EYFP and pGH19-mRFP recombinant vectors

[0044] 1. Experimental materials

[0045] Max DNA Polymerase Kit was purchased from TaKaRa Company, AxyPrep DNA Gel Recovery Kit was purchased from AXYGEN Company, EcoRI and XbaI Restriction Endonuclease Kit and T4 DNA Ligase Kit were purchased from Thermo Company, Trans-T1 Escherichia coli Competent Cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0046] 2. Experimental steps

[0047] 2.1 PCR amplification and recovery of EYFP and mRFP sequences

[0048] 1) Design amplification primers for EYFP and mRFP sequences, synthesized by Nanjing GenScript Biotechnology Co., Ltd., and purified by RPC.

[0049]

[0050] 2) Using TaKaRa's For Max DNA Polymerase Kit, add the following components to a 200μL PCR tube to configure a 25.0μL reaction system.

[0051]

[0052] 3) According to The Max DNA Polymerase kit manual sets the PCR amplification reaction program.

[005...

Embodiment 2

[0066] Example 2 Construction of honeybee (Apis mellifera) RDL gene expression vectors pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL

[0067] 1. Experimental materials

[0068] Reagent and restriction endonuclease HindIII were purchased from Thermo Company, PrimeScript TM The RT Kit with gDNA Erase was purchased from TaKaRa Company, the ClonExpress II One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd., and the rest of the experimental materials were the same as in Example 1.

[0069] 2. Experimental steps

[0070] 2.1 Cloning of base sequence of coding region of AmRDL gene

[0071] 1) Download the nucleotide sequence of the honeybee RDL gene (AmRDL) from the NCBI website, design primers to amplify its complete coding region sequence, synthesize it by Nanjing GenScript Biotechnology Co., Ltd., and purify it by RPC.

[0072]

[0073] 2) Using TaKaRa's For Max DNA Polymerase Kit, add the following components to a 200μL PCR tube to configure a 25.0μ...

Embodiment 3

[0091] Example 3 Expression and detection of AmRDL with EGFP or mRFP gene and gene without fluorescent tag in Xenopus oocytes

[0092] 1. Experimental materials

[0093] NotI restriction enzyme and mMESSAGE mMACHINE TM T7 kit was purchased from Thermo Company, phenol chloroform (phenol: chloroform: isoamyl alcohol = 25:24:1, pH>7.8) was purchased from Beijing Suo Lai Bao Technology Co., Ltd., Xenopus was purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences supply.

[0094] 2. Experimental steps

[0095] 2.1 Linearization of pGH19-AmRDL, pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL vectors

[0096] 1) The NotI restriction endonuclease kit from Thermo Company was used to digest the pGH19-AmRDL, pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL vectors. Add the following components to 200 μL PCR tubes respectively to configure a 150 μL reaction system:

[0097]

[0098] Mix the above reaction system evenly, then digest in a PCR machine at 37°C for 7.5h, hea...

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Abstract

The invention discloses a xenopus laevis oocyte expression carrier with a yellow or red fluorescence protein label and application. pGH19 plasmid is modified through a molecular biology means, and isspecifically developed to be a carrier pGH19-EYFP or pGH19-mRFP which is suitable for expression of the xenopus laevis oocyte and contains the enhanced yellow or monomer red fluorescence protein label. The carrier can normally express the target gene, and can detect the distribution situation and the expression mode of the target protein with the yellow or red fluorescence label through a fluorescence microscope or a confocal microscope, and thus a more visual and simpler detection method can be provided for the protein situation of xenopus laevis oocyte expression. The spatiotemporal dynamicsof the protein expression can be observed in real time. The expression situation of the target protein can be detected only by using an universal antibody as for the following extraction, purification, identification and functional analysis of the target protein, the experimental process is greatly simplified, and the experimental time is saved and economic cost is reduced.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering. Specifically, it relates to Xenopus laevis oocyte expression vectors with yellow or red fluorescent labels and their applications. Background technique [0002] Xenopus oocytes are a powerful and widely applicable heterologous expression system, which is widely used in exogenous calcium ion channels, chloride ion channels, potassium ion channels, sodium ion channels, γ-aminobutyric acid Expression of heterologous proteins such as receptors, glutamate receptors, acetylcholine receptors, and research on their physiological and pharmacological properties. It plays an important role in exploring the mechanism of action of compounds, evaluating and screening the efficacy and safety of target compounds, and is an indispensable heterologous expression in the fields of biology, medicine, neurology, and pesticides. system. [0003] Yellow and red fluorescent proteins refer to p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65
CPCC12N15/65C12N15/85C12N2800/106
Inventor 赵春青盛成旺贾忠强黄秋堂韩召军刘泽文
Owner NANJING AGRICULTURAL UNIVERSITY
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