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Construction methods and application of fusion protein and vector thereof

A fusion protein and protein technology, applied in the field of fusion proteins, can solve the problems of unknown TALEN transfection efficiency and cleavage activity, and achieve the effect of simple preparation method

Inactive Publication Date: 2015-04-08
广西即泰健康咨询有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is necessary to transfer TALEN plasmid pairs into cells through Lipo2000 or virus, but due to the lack of a control, the transfection efficiency and cleavage activity of TALEN are unknown

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A fusion protein structure provided by the present invention is EGFP-_mODC_NLS.

[0028] Its preparation method is:

[0029] The Escherichia coli BL21 strain with the recombinant plasmid EGFP-_mODC_NLS was expanded and cultured, and induced by IPTG overnight at 12-18°C, and the obtained EGFP-_mODC_NLS protein cells were collected, and the soluble supernatant was obtained by centrifugation after high-pressure crushing. Ni column affinity chromatography; after the target protein is combined with the Ni column, it is first washed with the rinsing solution, then washed with the elution buffer, and then replaced with a high-salt buffer in the eluted fraction with a desalting column containing 20% Glycerol in PBS solution, followed by SDS-PAGE detection, the obtained is the purified EGFP-_mODC_NLS protein.

[0030] The expression vector of a fusion protein provided by the present invention comprises an EGFP-_mODC_NLS fusion protein and a pair of TALEN plasmids cutting NLS; i...

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Abstract

The invention discloses construction methods and application of a fusion protein and a vector thereof and aims to provide a method for constructing a fusion protein scientifically and effectively by taking TALEN (transcription activator-like effector nuclease) as a cell strain. The method is technically characterized in that the fusion protein has a structure of EGFP-_mODC_NLS, and an expression vector of the fusion protein comprises the fusion protein with the structure of EGFP-_mODC_NLS and an NLS cutting TALEN plasmid pair of the fusion protein. The fusion protein is used for the NLS cutting TALEN plasmid pair and a prokaryotic expression protein with the structure of EGFP_mODC_NLS_TALEN_R and a protein with the structure of EGFP_mODC_NLS_TALEN_R, and the fusion protein can be used for verifying the transfection efficiency of the TALEN plasmid pair and the activity of the TALEN plasmid pair in cells. The invention belongs to the field of biotechnology.

Description

technical field [0001] The invention discloses a fusion protein, specifically, a fusion protein for verifying the transfection efficiency of a TALEN plasmid pair and its activity in cells and verifying the transfection efficiency of other plasmids. The invention also discloses The construction method and application of the fusion protein. Background technique [0002] The middle and C-terminal amino acids of mouse ornithine decarboxylase (MODC) contain two degradation domains of PEST sequence, which promote the degradation of the protein and make the half-life of the protein very short. This feature is important in gene function There are great applications in research. [0003] Transcription activator-like effector nuclease (TALEN) technology is a new molecular biology tool. TALEN technology has been widely used in gene modification. [0004] Especially the establishment of animal models and cell models. However, when making a cell model, or knocking out a certain gene ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/88C12N15/867
Inventor 霍依然李红梅覃启红黄龙
Owner 广西即泰健康咨询有限公司
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