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Histidine fluorescent probe as well as preparation method and application thereof

A fluorescent probe and histidine technology, applied in the field of bioengineering, can solve the problems of inability to detect cells in real time and accurately, and achieve high-throughput compound screening, large dynamic changes in fluorescence, and good specificity

Active Publication Date: 2017-06-30
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

The common detection method of histidine is capillary electrophoresis (Li X-t et al., Chem Res Chin Univ 2013,29(3):434-438; Meng J et al., The Analyst 2010,135(7):1592-1599), highly efficient Liquid chromatography (Tateda N etc., Analytical sciences: the international journal of the Japan Society for Analytical Chemistry 2001,17(6):775-778; Wadud S etc., Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2002, 767(2):369-374), UV-visible spectrophotometry (Hortala MA et al., J Am Chem Soc 2003,125(1):20-21; PuF et al., Anal Chem 2010,82(19):8211-8216 ; Du J et al., Chemical communications (Cambridge, England) 2013,49(47):5399-5401; Engeser M et al., Chemical Communications 1999, (13):1191-1192) and fluorescence spectrometry (Engeser M et al., Chemical Communications 1999, (13): 1191-1192), but these detection methods have great defects in the study of live cells, which require time-consuming sample processing: cell disruption, separation, extraction and purification, etc., and cannot be used for real-time analysis of intact cells. accurate detection

Method used

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  • Histidine fluorescent probe as well as preparation method and application thereof
  • Histidine fluorescent probe as well as preparation method and application thereof
  • Histidine fluorescent probe as well as preparation method and application thereof

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preparation example Construction

[0039] The present invention also provides a preparation method for the above-mentioned histidine fluorescent probe, comprising the following steps: 1) linking the nucleotide sequence encoding the B1-A-B2 type probe with the pRSETb vector to obtain recombinant expression in Escherichia coli 2) transferring the recombinant expression vector of Escherichia coli into host cells; 3) cultivating the host cells and isolating the histidine fluorescent probe.

[0040] In the present invention, the synthesis of the nucleotide sequence encoding the B1-A-B2 type probe adopts the method of PCR amplification or artificial synthesis. When the PCR amplification method is used, primers are designed according to the nucleotide sequence described in the present invention, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art is used as a template to amplify. When the nucleotide sequence is larger than 2500bp, it is prefer...

Embodiment 1

[0120] Construction of pRSETb-HBP plasmid

[0121] The HisJ gene in the Escherichia coli gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and HindIII, and the pRSETb vector was subjected to the same double digestion. After ligation with T4 DNAligase, the ligation product was transformed into MachI, and the transformed MachI was spread on LB plates (ampicillin 100ug / mL), and cultured at 37°C overnight. After plasmid extraction of the growing MachI transformants, PCR identification was performed. After the positive plasmid is correctly sequenced, the subsequent plasmid construction is carried out.

[0122] The primer sequences for constructing the pRSETb-HBP plasmid are shown in Seq ID NO.39-40.

[0123] Plasmid construction and detection of different insertion sites of pRSETb-HBP-cpFP fluorescent probe

[0124] In this example, we selected 89 / 90, 89 / 91, 89 / 92, 89 / 93, 90 / 91, 90 / 92, 90 / 93, 91 / 92 based on the HBP cry...

Embodiment 2

[0132] According to the method in Example 1, cpYFP was replaced by blue fluorescent protein cpBFP, which was fused into HBP to construct a histidine blue fluorescent protein fluorescent probe. The result is as image 3 As shown, the results of fluorescence detection showed that 90 / 91, 186 / 191, 187 / 191, 189 / 190, 189 / 191, 190 / 191, and 190 / 193 responded more than 3 times to histidine.

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Abstract

The invention provides a histidine fluorescent probe. The histidine fluorescent probe comprises polypeptide B sensitive to histidine and fluorescent protein A performing expression on histidine, wherein the fluorescent protein A in inserted into the polypeptide B to divide the polypeptide B into two parts of B1 and B2, and a B1-A-B2 type probe structure is formed; the polypeptide B and the histidine interact to enable fluorescent signals of the fluorescent protein A to be changed; and the polypeptide B is HBP and a mutant thereof. The histidine fluorescent probe provided by the invention is relatively small in albumen molecular weight, is easy to mature, large in fluorescent dynamic changes and good in specificity, and can be expressed in different subcells of cells by a gene manipulation method, and histidine can be detected inside and outside the cells in a high-flux and quantitative manner.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a histidine fluorescent probe and its preparation method and application. Background technique [0002] Histidine is one of the 20 basic amino acids and is an important amino acid. Since the pKa of its side chain is 6.0 (Nelson DL et al., Germany: Springer 2009), its side chain can be deprotonated in a neutral environment The state becomes the protonated state. Based on this property, histidine residues can act as both proton acceptors and proton donors in many intracellular reactions (Rebek J et al., Struct Chem 1:129–131 1990; Polgár L. et al., CellMol Life Sci 62:2161–2172 2005). Moreover, it contains a highly reactive imidazole ring, which plays an important role in the transport of metal ions in biological systems (Creighton TE. et al., The encyclopedia of molecular biology 1999; Kusakari Y et al., Curr Eye Res[J] 1997, :600-604.). Therefore, the selective determi...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N15/70G01N21/64
CPCC07K14/00C12N15/70G01N21/6402
Inventor 杨弋赵玉政胡晗阳顾燕芳徐磊陶荣坤
Owner EAST CHINA UNIV OF SCI & TECH
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