Fusion protein and application thereof

A fusion protein and protein technology, which is applied in the field of biomedicine, can solve the problems of the influence of induction results, time-consuming, unfavorable PI3K/Akt pathway control, etc., and achieve the effect of rapid activation and regulation and high spatial resolution

Active Publication Date: 2020-07-07
SHENZHEN INST OF ADVANCED TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is simpler than the traditional chemical small molecule induction method, and has higher temporal and spatial resolution, but it needs to express two fusion proteins in cells, namely CIBN-CAAX and mCh-CRY2-iSH2, which are required for each protein to achieve the best effect. It takes a lot of time and energy to explore the expression level, and iSH2 is derived from the PI3K subunit, overexpression may affect the induction result, which is not conducive to the control of the PI3K / Akt pathway

Method used

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  • Fusion protein and application thereof
  • Fusion protein and application thereof
  • Fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Preparation of embodiment 1lyn-Dab1-Cry2olig-mCh fusion protein

[0042] Using the basic technology of molecular cloning, the DNA sequences encoding the membrane localization sequence of lyn protein, Dab1 protein, light-sensitive protein Cry2olig and red fluorescent protein mCh (mCherry) were connected, introduced into the expression vector pCAG-DsRed2, and the recombinant expression vector was obtained and transformed into Host bacteria Escherichia coli, induced expression to obtain the fusion protein (see the schematic diagram of the structure figure 2 ), has the amino acid sequence shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO.6.

Embodiment 2

[0043] Example 2 Preparation of lyn-Dab1-Cry2olig-mCit fusion protein

[0044] Using the basic technology of molecular cloning, the DNA sequences encoding the membrane localization sequence of lyn protein, Dab1 protein, light-sensitive protein Cry2olig and yellow fluorescent protein mCit (mCit) were connected, and introduced into the expression vector pCAG-DsRed2 to obtain the recombinant expression vector, which was transformed into The host strain Escherichia coli is induced to express the fusion protein, which has the amino acid sequence shown in SEQ ID NO:4 and the nucleotide sequence shown in SEQ ID NO.7.

Embodiment 3Dab

[0045] Example 3 Preparation of Dab1-Cry2olig-mCit-KRas fusion protein

[0046] Using the basic technology of molecular cloning, the DNA sequences encoding Dab1 protein, light-sensitive protein Cry2olig, yellow fluorescent protein mCit (mCit) and KRas protein membrane localization sequence were connected and introduced into the expression vector pCAG-DsRed2 to obtain a recombinant expression vector, which was transformed into The host strain Escherichia coli is induced to express the fusion protein, which has the amino acid sequence shown in SEQ ID NO:5 and the nucleotide sequence shown in SEQ ID NO.8.

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Abstract

The invention relates to the technical field of biological pharmaceuticals, in particular to a fusion protein and an application thereof. According to the fusion protein, a plasma membrane positioningsequence, a Dab1 protein, a light-sensitive protein and a fluorescent protein are in fusion to be positioned to a lipid raft region or a non lipid raft region of a cytoplasmic membrane, and through illumination, quick activation and adjustment and control of a PI3K / Akt signal channel can be realized locally at a high time and space resolution. The fusion protein has great important significance in quick effective specific opening and closing control of the signal channel, contributing to understanding a cascade transmission rule of signals and researching diseases closely related to the signal channel.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a fusion protein and its application. Background technique [0002] Phosphoinositide 3-kinase (PI3K) is an intracellular phosphatidylinositol kinase that accepts signals from tyrosine kinases and G protein-coupled receptors and releases substrates at the plasma membrane. The small molecule Ptd Ins(4,5)P2 catalyzes the formation of Ptd Ins(3,4,5)P3 (hereinafter referred to as PI3P). PI3P activates downstream proteins such as Akt. This signaling pathway widely exists in various cells and is an important pro-survival signaling pathway in cells, regulating various activities such as cell proliferation, differentiation, growth, survival, metabolism and migration. Studies have shown that abnormal PI3K / Akt pathway is closely related to the occurrence and development of human tumors, neurodegenerative diseases, insulin resistance-related diseases such as type II diabetes, and cardi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/13C12N15/70C12N1/21A61K38/16A61P25/28A61P9/00A61P9/10A61P3/10
CPCC07K14/415C07K14/43595C12N15/70A61P25/28A61P9/00A61P9/10A61P3/10C07K2319/60A61K38/00Y02A50/30
Inventor 储军王亮
Owner SHENZHEN INST OF ADVANCED TECH
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