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A kind of expression vector and its application for the study of bimolecular fluorescence complementation

An expression vector and bimolecular technology, which can be used in the introduction of foreign genetic material, material inspection products, recombinant DNA technology, etc. using vectors. It can solve problems such as high price and achieve high flexibility.

Active Publication Date: 2018-07-17
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Gateway cloning requires the use of commercial kits, which are very expensive, and Gateway cloning requires two cloning steps of cloning the gene into the entry vector and the target vector

Method used

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  • A kind of expression vector and its application for the study of bimolecular fluorescence complementation
  • A kind of expression vector and its application for the study of bimolecular fluorescence complementation
  • A kind of expression vector and its application for the study of bimolecular fluorescence complementation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of a new expression vector pBiFC-GG for bimolecular fluorescence complementation research

[0051] 1) Transform the multiple cloning sites of the traditional BiFC vectors pSAT-cEYFP-C1, pSAT-nEYFP-C1, pSAT-cEYFP-N1 and pSAT-nEYFP-N1, respectively introduce the GG cloning frame structure, and transform the vectors respectively Named pSAT-cC1-GG, pSAT-nC1-GG, pSAT-cN1-GG and pSAT-nN1-GG;

[0052] Wherein, the GG cloning frame structure includes BsaI restriction site, SfiI restriction site, ccdB gene, SfiI restriction site, BsaI restriction site connected in sequence, the nucleosides of the GG cloning frame structure The acid sequence is shown in SEQUENCE NO.1. Schematic diagram of the structure of the GG cloning cassette figure 1 As shown, the wavy line represents the enzyme cleavage site.

[0053] Specifically, synthesize the primer pair CccdBF: gagacctaggttctgggccaggatggcc attctcgactaagttggcag and CccdBR: aaccacctgagaccgaaggcccagatggcc ctgtgtat...

Embodiment 2

[0063] Example 2 Using pBiFC-GG-N1 to study the interaction between papaya eIF4E and PRSV-Vpg

[0064] The inventor's laboratory team has used yeast two-hybrid technology to find that papaya eIF4E interacts with papaya ringspot virus PRSV-Vp, so these two proteins are selected to verify the reliability of the pBiFC-GG-N1 vector in the present invention sex.

[0065] 1. Amplification of papaya eIF4E and PRSV-Vpg genes

[0066] Two pairs of primers were designed to amplify the papaya eIF4E gene, Golden-4EF: aca ggtctcagtctatggtagtagaaggaaccc and Golden-4ER: aca ggtctcacacc taccgagtagcgattcttag, and Gibson-4EF: acctagttctgggccagga atggtagtagaaggaaccc and Gibson-4ER: cctgagaccgaaggccGoldcagagattgccg for the primer pair For Golden Gate cloning, the primer pair Gibson-4E was used for closed-circle Gibson splice cloning.

[0067]设计两对扩增番木瓜环斑病毒PRSV-Vpg基因的引物,Golden-VpgF:acaggtctcatggc atgggtttctccgcgcgacag和Golden-VpgR:aca ggtctcactgattcatggtgaacagattttgtt,以及Gibson-VpgF:gagaccatggagggc...

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Abstract

The invention provides a novel expression vector for bimolecular fluorescence complementation (BiFC) research. Expression cassettes of 2 segments of a fluorescent protein are integrated onto the vector; and gene sequences of 2 target proteins can be simultaneously cloned to the vector by one-step reaction (Golden Gate cloning or closed-loop Gibson splicing process) and respectively form fusion expression with the 2 segments of the fluorescent protein. Therefore, when the vector is used for BiFC research, only one vector is needed and introduced into cells, thereby greatly facilitating the operation of the plant BiFC experiment. The vector is compatible with the two novel cloning methods of Golden Gate cloning or closed-loop Gibson splicing. The vector is a high-copy plant expression vector containing T-DNA sequence, and can be simultaneously used for instantaneous and stable expression of plants.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to an expression carrier used for bimolecular fluorescence complementation research and application thereof. Background technique [0002] Bimolecular fluorescence complementation (BiFC) technology, first reported in 2002, is a new technology for intuitively and rapidly determining the localization and interaction of target proteins in living cells. This technology skillfully fuses two complementary fragments of the fluorescent protein molecule with the target protein, and if the activity of the fluorescent protein is restored, it indicates that the two target proteins have interacted. If there is no interaction between the 2 target proteins, they cannot be excited to fluoresce. [0003] At present, various BiFC systems have been widely used for the interaction of various proteins, and have been applied in various systems such as Escherichia coli, fungi, yeast, mamma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/66G01N33/68
Inventor 言普周鹏沈文涛庹德财黎小瑛
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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