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Fluorescent protein for copper ion detection and its encoding nucleotide sequence and application

A technology of nucleotide sequence and fluorescent protein, applied in application, measurement device, biological test, etc., can solve problems such as endangering human health, and achieve the effect of alleviating loss of effectiveness and strong salt tolerance

Active Publication Date: 2021-02-23
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Copper has important biological functions, but excess will seriously endanger human health

Method used

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  • Fluorescent protein for copper ion detection and its encoding nucleotide sequence and application
  • Fluorescent protein for copper ion detection and its encoding nucleotide sequence and application
  • Fluorescent protein for copper ion detection and its encoding nucleotide sequence and application

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preparation example Construction

[0055] According to one aspect of the present invention, a kind of above-mentioned Cu 2+ A method for preparing a fluorescent protein for detection, the preparation method comprising the following steps:

[0056] Firstly, the recombinant co-expression vector will be used to encode the above Cu 2+ The nucleotide sequence of the detection fluorescent protein and the chromogenic substrate nucleotide sequence are transferred to the host cell to induce expression, and Cu 2+ Fluorescent proteins for detection.

[0057] Cu provided by the present invention 2+ A method for preparing a fluorescent protein for detection, the preparation method encodes the above-mentioned Cu 2+ The nucleotide sequence of the detection fluorescent protein and the chromogenic substrate nucleotide sequence are transferred to the host cell to induce expression, and Cu 2+ Fluorescent proteins for detection. This preparation method can produce Cu on a large scale 2+ Fluorescent protein for detection has ...

Embodiment 1

[0069] The acquisition of embodiment 1 nucleotide sequence:

[0070] Using PCR technology, design primers to amplify the DNA fragment of cyanobacterial phytochrome SPI1085g2 from S. subsalsa FACHB351;

[0071] The upstream primer sequence is:

[0072] 5'-CTAGAGCTCTTCCCTCGAAATTGAGCAGATTTTCC-3';

[0073] The downstream primer sequences are:

[0074] 5-GTTAAGCTTAGCGGGTTTTGGCTAATAATTCGGC-3'.

[0075] Restriction sites SacI and HindⅢ were designed on the upstream and downstream primers respectively. The PCR product was digested with SacI and HindⅢ to obtain a purified DNA fragment, which was ligated with the vector pETDuet-1 digested with SacI and HindⅢ to construct the expression vector pETDuet-SPI1085g2. Sequencing of SPI1085g2 to obtain the coded Cu 2+ Nucleotide sequences of fluorescent proteins for detection.

Embodiment 2

[0076] The construction of embodiment 2 recombinant co-expression vectors:

[0077] Such as figure 1 As shown, the heme oxidase gene ho1 and the biliverdin reductase gene pebS were fused, constructed into the second multiple cloning site of the expression vector pETDuet-SPI1085g2 through Bgl II and XhoI, and finally constructed to contain Cu 2+ The detection uses the nucleotide sequence of the fluorescent protein and the recombinant co-expression vector pETDuet-SPI1085g2-ho1:pebS of the chromogenic substrate nucleotide sequence.

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Abstract

The invention provides a fluorescent protein for copper ion detection and its encoding nucleotide sequence and application, and relates to the technical field of molecular biology. The Cu 2+ The fluorescent protein for detection includes the amino acid sequence shown in Seq No.01. The protein can efficiently bind phycoerythrin PEB and emit strong orange fluorescence, while the above Cu 2+ The fluorescent protein for detection has a specific response to copper ions, and can be selectively quenched by copper ions, but basically has no effect on other ions. In addition, the Cu 2+ The fluorescent protein for detection also has strong salt tolerance, which effectively alleviates the problem that the existing fluorescent protein for copper ion detection loses its effectiveness under high salt concentration.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a fluorescent protein for copper ion detection, its coding nucleotide sequence and application. Background technique [0002] The detection and quantification of heavy metals is very important for environmental monitoring and bioclinical toxicology testing. In the prior art, certain fluorescent proteins bind heavy metals at a certain site of the protein to change the microenvironment of the chromophore, resulting in the effect of fluorescence quenching, and then used as biosensors for heavy metals. Within a certain range, fluorescent proteins The fluorescence intensity depends on the surrounding heavy metal concentration. Therefore, the relationship between the heavy metal and the fluorescence intensity of the fluorescent protein can be established, and the quenching effect of the fluorescence intensity can be used to reflect the concentration of the heavy metal. [0...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/70C12N15/53C12N1/21G01N33/68G01N33/58C12R1/19
CPCC07K14/195C12N9/001C12N15/70C12Y103/07006G01N33/582G01N33/68G01N2333/195
Inventor 伍贤军李萍萍朱咏莉盛怡蒋苏丹
Owner NANJING FORESTRY UNIV