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Green fluorescent protein Clover4, probe derived from green fluorescent protein Clover4 and based on bioluminescence resonance energy transfer and application of green fluorescent protein Clover4

A green fluorescent protein and resonance energy transfer technology, applied in the field of BRET serum antibody detection system, can solve the problems of high brightness and high detection sensitivity, and achieve the effect of early diagnosis

Active Publication Date: 2021-08-03
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to jellyfish green fluorescent protein, the green fluorescent protein mNeonGreen from amphioxus (Shaner NC, L.G., Chammas A, Ni Y, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M, Davidson MW, Wang J.A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10(5), 407-409(2013).), its brightness is a little higher than that of Clover, both of which are the green fluorescent proteins with the highest brightness at present, but still cannot meet the high detection requirements need for sensitivity

Method used

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  • Green fluorescent protein Clover4, probe derived from green fluorescent protein Clover4 and based on bioluminescence resonance energy transfer and application of green fluorescent protein Clover4
  • Green fluorescent protein Clover4, probe derived from green fluorescent protein Clover4 and based on bioluminescence resonance energy transfer and application of green fluorescent protein Clover4
  • Green fluorescent protein Clover4, probe derived from green fluorescent protein Clover4 and based on bioluminescence resonance energy transfer and application of green fluorescent protein Clover4

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Experimental program
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Effect test

Embodiment 1

[0059] Example 1 Construction of green fluorescent protein Clover4 and detection of photophysical properties

[0060] (1) Analysis of the crystal structure of the green fluorescent protein Clover ( figure 1 b), and compared with the homologous sequence, rationally design and site-directed mutation the key sites that affect the spectrum of the fluorescent protein and the amino acids that interact with it, and then express and screen the mutants on the constitutive expression vector pNCS, used The expression strain is Stellar. In order to ensure the integrity of the library, 10 clones were set up for each mutant. Finally, the fluorescent properties of the mutant were detected by naked eye and blue LED excitation light through an orange acrylic filter, and a single clone expressing a fluorescent protein with a blue-shifted spectrum was screened out. clone. Subsequently, the non-conserved amino acid residues around the luminescent group of the mutant fluorescent protein with a b...

Embodiment 2

[0068] Example 2 Application of Clover4 in BRET and serum antibody detection Clover4-LUMABS system (mNeonGreen is abbreviated as mNeonG in the probe)

[0069] (1) Clover4-LUMABS-HIV probe

[0070] Firstly, mNenG-LUMABS-HIV and Clover4-LUMABS-HIV probes were respectively constructed, and the schematic diagrams of the probe structures are shown in Figure 4 As shown, mNenG-LUMABS-HIV is a probe that connects luciferase (Nluc) and mNeonGreen through a sequence containing an HIV epitope, and the probe also includes a SH3 / sp1 domain, while Clover4-LUMABS-HIV is a probe that contains The sequence of the HIV epitope is connected to the probe of luciferase (Nluc) and Clover4, and the probe also includes the SH3 / sp1 domain. See Table 2 for antigenic epitopes.

[0071] Tested in fetal calf serum (FBS), the test results are shown in Table 3. The experimental results show that the BRET efficiency (hiBRET) of the Clover4-LUMABS-HIV probe is 4.6 times that of mNenG-LUMABS-HIV in the absen...

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Abstract

The invention relates to a green fluorescent protein Clover4, a probe derived from green fluorescent protein Clover4 and based on bioluminescence resonance energy transfer and application of green fluorescent protein Clover4 In particular, the invention discloses a green fluorescent protein Clover 4, and compared with an amino acid sequence of Clover, an amino acid sequence of the green fluorescent protein has 11 mutation sites. The invention discloses a protein pair containing the green fluorescent protein and used for bioluminescence resonance energy transfer detection and a probe based on bioluminescence resonance energy transfer. After the green fluorescent protein is used for a bioluminescence resonance energy transfer probe, the detection limit is reduced, the detection sensitivity is improved, the detection dynamic range is widened, an antibody can be detected in a lower-concentration antibody environment, and early diagnosis is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection system for green fluorescent protein Clover4 and its derived BRET serum antibody. Background technique [0002] Bioluminescence Resonance Energy Transfer Technology [0003] Bioluminescence has become one of the important means of in vivo optical imaging because it does not require light excitation and has low background. However, luciferase (which generates photons by catalyzing the biochemical reaction of the substrate) not only has a low quantum yield, but also emits photons at a shorter wavelength, thus limiting its application in animals. The bioluminescent resonance energy transfer (BRET) technology based on luciferase and fluorescent protein can solve the above problems well. Bioluminescence Resonance Energy Transfer (BRET) is an efficient optical "molecular ruler". Its technical principle is based on non-radiative energy transfer. The process of tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12G01N33/68G01N33/58G01N33/569G01N33/576
CPCC07K14/43595G01N33/6854G01N33/582G01N33/581G01N33/56983G01N33/56994G01N33/5761G01N2333/11G01N2333/185G01N2333/165G01N2469/20
Inventor 储军邓梦颖袁静金宗文
Owner SHENZHEN INST OF ADVANCED TECH
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