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Preparation method and application of 3*FLAG tag fusion expression vector

An expression vector and tag fusion technology, applied in the field of molecular biology, can solve the problems of low transformation efficiency, application obstacles, long period of tobacco seedling breeding, etc., and achieve the effect of increasing sensitivity and increasing binding.

Pending Publication Date: 2022-03-08
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this method can be used to obtain a large amount of "target gene-FLAG" fusion protein, the tobacco seedling cultivation cycle is long, and the transformation steps mediated by Agrobacterium are complicated.
In addition, the tobacco system cannot simulate the environment of different plants, and the research on protein function and protein interaction, especially when using the "target gene-FLAG" fusion protein carrier system to find the interaction protein of the target gene in plants, requires the "purpose Gene-FLAG" fusion vector should be expressed in the target plant under study, at this time, the application of Agrobacterium-mediated transformation methods in other plants such as Arabidopsis, rice, and maize is hindered
[0004] PEG-mediated transformation of plant protoplasts is currently the most widely used fusion vector expression system, but the pCambia1300 framework is too large, the transformation efficiency is low, and the copy number of the pCambia1300 framework is low, which cannot meet the large number of plasmid requirements of the protoplast transformation system

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  • Preparation method and application of 3*FLAG tag fusion expression vector
  • Preparation method and application of 3*FLAG tag fusion expression vector
  • Preparation method and application of 3*FLAG tag fusion expression vector

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Experimental program
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Effect test

Embodiment 1

[0048] The preparation method of the 3×FLAG tag fusion expression vector of this embodiment, the method is:

[0049] S1. Digest the pGL3 Basic vector with KpnI / XhoI (the physical map of the pGL3 Basic vector is figure 1 ), after reacting at a temperature of 37° C. for 6 h, the digested product was separated with 1% agarose gel, and the band with a size of 4.8 Kb was cut out, and the product was recovered with a DNA gel recovery kit to obtain KpnI / Recovered product of XhoI double digestion of pGL3 Basic vector; enzyme digestion system of KpnI / XhoI double digestion of pGL3 Basic vector is: KpnI enzyme 1 μL, XhoI enzyme 1 μL, Cutsmartbuffer 5 μL, pGL3 Basic vector 10 μL, sterilized ultrapure water to 50 μL ;

[0050] S2, using the pRGEB32Bar-Cas9 plasmid (MK791524.1) as a template, and using specific primers F1 and specific primers R1 as primers to perform a PCR reaction to clone the 2×35S promoter, after PCR amplification, the PCR amplification product was washed with 1% Sepa...

Embodiment 2

[0065] This example is the application of the 3×FLAG tag fusion expression vector pProto-3×FLAG prepared in Example 1. The 3×FLAG tag fusion expression vector pProto-3×FLAG is used for the transformation of plant protoplasts; for the target protein Expression in plant protoplasts; said plant protoplasts include Arabidopsis protoplasts and maize protoplasts.

[0066] (1) Cloning enhanced yellow fluorescent protein (YFP), connecting YFP to the XhoI / HindIII of the 3×FLAG tag fusion expression vector pProto-3×FLAG prepared in Example 1 by homologous recombination to obtain pProto-YFP- 3×FLAG fusion expression vector.

[0067] The pProto-YFP-3×FLAG fusion expression vector was transformed into maize protoplasts by PEG-mediated method, and the YFP-3×FLAG fusion protein was expressed in large quantities.

[0068] Observe the luminescent situation of the transformed maize protoplasts under the 488nm excitation light of the laser confocal microscope, as shown in image 3 , basically ...

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Abstract

The invention provides a preparation method of a 3 * FLAG tag fusion expression vector, which comprises the following steps: using a KpnI / XhoI double-enzyme digestion pGL3 Basic vector to obtain a recovered product of the KpnI / XhoI double-enzyme digestion pGL3 Basic vector, using a pRGEB32Bar-Cas9 plasmid as a template to obtain a 2 * 35S promoter PCR recovered product, carrying out a ligation reaction to obtain an intermediate vector pGL-35S, carrying out double-enzyme digestion, connecting an MCS annealing primer to obtain an intermediate vector pGL-35S-MCS, carrying out double-enzyme digestion, connecting a Nos Ter sequence to obtain the 3 * FLAG tag fusion expression vector. The preparation method comprises the following steps: firstly, performing double enzyme digestion on an intermediate vector pGL-35S-MCS-Nos to obtain the intermediate vector pGL-35S-MCS-Nos, connecting a connecting peptide Linker primer subjected to denaturation annealing after double enzyme digestion to obtain the intermediate vector pGL-35S-MCS-Linker-Nos, and connecting a 3 * FLAG tag annealing primer after double enzyme digestion to obtain the 3 * FLAG tag fusion expression vector. The 3 * FLAG tag fusion expression vector prepared in the invention has a small framework, has a full length of 4088bp, and is suitable for plant protoplast transformation.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a preparation method and application of a 3×FLAG tag fusion expression vector. Background technique [0002] FLAG is a polypeptide consisting of 8 amino acid residues (N-DYKDDDDK-C), about 1012Da. In the research of protein expression, protein interaction, etc., the target gene to be studied can be connected with the FLAG polypeptide sequence through genetic engineering technology. The FLAG polypeptide can be connected to the C-terminal or N-terminal of the target protein, and then the "target gene- The expression vector fused with FLAG" is transformed into bacteria, yeast, animal or plant cells. On the one hand, because the molecular weight of the FLAG tag is very small, it will not cover the protein epitope and structural domain in the fusion protein, and it is not easy to change the function, localization, secretion and transportation of the fusion prote...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/82C12N5/10
CPCC07K14/415C12N15/82C07K2319/43
Inventor 徐玉芳姚文李涛张会勇林楠孙玉慧连红梅贾利华李阳
Owner HENAN AGRICULTURAL UNIVERSITY
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