Improved bimolecular fluorescence complementation carrier for researching protein interaction

A bi-molecular and binary vector technology, applied in the field of genetic engineering, can solve the problems of cumbersome, difficult enzyme cutting and connection, and complicated process.

Inactive Publication Date: 2012-11-07
ZHEJIANG NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has some disadvantages: First, rare restriction endonucleases are relatively expensive, and it is not easy to digest and connect, which will greatly increase the difficulty in the research process
Secondly, it is connected to the intermediate vector and the binary expression vector successively. This process is relatively complicated and cumbersome.
Another method is to use the GATEWAY carrier. Although this method is fast and efficient, the reagents used are expensive

Method used

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  • Improved bimolecular fluorescence complementation carrier for researching protein interaction
  • Improved bimolecular fluorescence complementation carrier for researching protein interaction
  • Improved bimolecular fluorescence complementation carrier for researching protein interaction

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Embodiment Construction

[0031] In a specific mode of the present invention, use pCAMBIA1301 (pCAMBIA1301 carrier map, see image 3 ) as the backbone, by eliminating the enzyme cutting sites (Ban II, Sac I, Kpn I, Sma I, BamH I, Sal I, Pst I, Sph 1), at the same time, steps such as adding CaMV35S terminator, cEYFP box or nEYFP box at the MCS place that eliminates restriction site, build a convenient, cheap operation platform. To study some proteins that may interact, just clone the gene corresponding to the target protein into the MCS of the above two binary expression vectors, then transform Agrobacterium, and inject the Agrobacterium containing the target binary expression vector into Nicotiana benthamiana , Fluorescence confocal microscopy to observe whether there is fluorescence, and then the interaction of the target protein can be obtained. See the specific technology roadmap image 3 .

[0032] Specifically, the following methods are used to obtain:

[0033] Part 1: Construction of pCAMBIA1...

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Abstract

The invention provides an improved bimolecular fluorescence complementation carrier for researching the protein interaction. The carrier includes a CaMV 35S promoter and a CaMV 35S terminator, and a cEYFP box or a nEYEP box is included between the CaMV 35S promoter and the CaMV 35S terminator. The construction cost of the carrier is low, while the construction efficiency is high.

Description

technical field [0001] The invention relates to a carrier and a method for preparing the carrier, belonging to the technical field of genetic engineering. Background technique [0002] Protein interaction is a basic physiological function in cells, which occurs in every physiological process in all subcellular structures and organelles. Clarifying when and where protein interaction occurs and how protein complexes are formed can provide a basis for determining Protein biological roles provide decisive clues. [0003] Bimolecular fluorescence complementation (BiFC) is a technology that uses the luminescent principle of fluorescent proteins to study protein interactions. The main process is to cut fluorescent proteins into two parts, the N-terminal and the C-terminal, and link them to the target protein respectively. The recombinant vector co-transfects cells. If there is an interaction between the target proteins, the N-terminal fragment and the C-terminal fragment of the fl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66
Inventor 王长春冯彩芬杨莉宋凯赵奕铭王伟杨玲俞盼盼李茶姿王锋青
Owner ZHEJIANG NORMAL UNIVERSITY
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