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Cyclic diguanylate optical probe as well as preparation method and application thereof

An optically active, nucleic acid molecule technology, applied in the field of optical probes, can solve the problems of cumbersome detection methods, easy interference of spectral overlap, poor water solubility, etc., and achieves the effect of eliminating sample processing steps, large dynamic changes in fluorescence, and good specificity.

Pending Publication Date: 2021-12-21
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

FRET-type probes include two fluorescent proteins, and there are problems such as the spectral overlap of the two proteins is easy to interfere, the efficiency of co-expression is low, and the water solubility is poor.
In the BRET method, luciferase Rluc requires exogenous addition of substrates to generate fluorescent signals, and the detection method is cumbersome.
In addition, c-di-GMP analogues GTP, GDP, ATP, AMP, ADP, NAD widely exist in vivo and in vitro + 、NADP + Seriously affect the accuracy of detection methods in the prior art

Method used

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  • Cyclic diguanylate optical probe as well as preparation method and application thereof
  • Cyclic diguanylate optical probe as well as preparation method and application thereof
  • Cyclic diguanylate optical probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] Example 1: c-di-GMP binding protein particles

[0156] The BldD gene in the Streptomyces coelicolor gene was amplified by PCR, the PCR product was purified and recovered after gel electrophoresis, and the pCDFduet vector was subjected to PCR amplification at the same time. Homologous recombination was used for ligation, and the ligation product was transformed into DH5α (TransGen Biotech) competent cells. The transformed DH5α was spread on LB plates (streptomycin 100ug / mL), and cultured at 37°C overnight. The growing DH5α transformant was extracted from the plasmid and then identified by PCR. After the positive plasmid is correctly sequenced, the subsequent plasmid construction is carried out.

Embodiment 2

[0157] Example 2: Expression and detection of cpYFP optical probes with different insertion sites and tandem linkers

[0158] In this example, the following sites were first selected to be inserted into cpYFP based on pCDFduet-BldD to obtain the corresponding plasmids containing the coding sequence of the singlet optical probe: 9 / 10, 10 / 11, 11 / 12, 16 / 17, 17 / 18, 18 / 19, 35 / 36, 36 / 37, 36 / 38, 36 / 39, 36 / 40, 36 / 41, 36 / 42, 37 / 38, 37 / 39, 37 / 40, 37 / 41 , 37 / 42, 38 / 39, 38 / 40, 38 / 41, 38 / 42, 39 / 40, 39 / 41, 39 / 42, 40 / 41, 40 / 42, 41 / 42, 42 / 43, 43 / 44, 44 / 45, 45 / 46, 46 / 47, 47 / 48, 48 / 49, 49 / 50, 50 / 51. The amino acid sequences of exemplary intercalating optical probes are shown in Table 1. The nucleic acid sequence of an exemplary insertion-type optical probe is shown in SEQ ID NO: 14 (BldD-36 / 41-cpYFP).

[0159] Table 1

[0160] sequence insertion site SEQ ID NO:6 36 / 37 SEQ ID NO:7 36 / 38 SEQ ID NO:8 36 / 39 SEQ ID NO:9 36 / 40 SEQ ID NO:10 36 / 41 SE...

Embodiment 3

[0169] Example 3, Expression and detection of cpGFP optical probes with different insertion sites and tandem linkers

[0170] According to the method in Example 1, cpYFP was replaced by green fluorescent protein cpGFP, fused to c-di-GMP binding protein to construct c-di-GMP green fluorescent protein fluorescent probe, expressed and detected according to the method in Example 2. The result is as image 3 , As shown in A, the results of fluorescence detection showed that 37 / 38, 37 / 40, 37 / 41, and 39 / 40 sites responded more than 2 times to c-di-GMP.

[0171] According to the method in Example 2, cpGFP multiplex optical probes containing different tandem linkers were obtained. The result is as image 3 , as shown in B, the test results show that the response to c-di-GMP exceeds 1 times: G02, G03, G04, G06, G07, G12, G13, G14, G15, G16, G17, G22, G23, G24, G26, G27, G32, G33, G34, G35, G36, G37, G42, G43, G44, G45, G46, G47, G52, G53, G54, G55, G56, G57; the response to c-di-GMP ...

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Abstract

The invention provides a c-di-GMP fluorescent probe, which comprises (A) an optically active polypeptide or a variant having at least 70% sequence identity and optical activity with the optically active polypeptide, and (B) a c-di-GMP sensitive polypeptide or a variant having at least 70% sequence identity and c-di-GMP sensitivity with the c-di-GMP sensitive polypeptide, where A is located within the sequence of B, which divides B into two parts B1 and B2, a structure with B1-A-B2 in the direction from the amino terminal to the carboxyl terminal is formed, or one or more A is located between two or more B and is connected with the B at the two ends through a connector. The c-di-GMP fluorescent probe provided by the invention is relatively small in protein molecular weight, easy to express, large in fluorescence dynamic change and good in specificity, related application research can be carried out in cells and escherichia coli through gene operation, and high-throughput, implementation and quantitative detection of c-di-GMP can be realized.

Description

technical field [0001] The invention relates to the technical field of optical probes, in particular to a cyclic diguanylic acid (c-di-GMP) optical probe and its preparation method and application. Background technique [0002] C-di-GMP (cyclic diguanylic acid) was originally discovered in 1987 by Moshe Benziman and colleagues who found that c-di-GMP can allosterically activate cellulose synthesis in α-staphylococcus proteus. c-di-GMP has been shown to regulate bacterial biofilm formation, bacterial motility, bacterial virulence, cell cycle, differentiation, and other processes. In addition, c-di-GMP can also function as a riboswitch. Modulating the c-di-GMP signaling pathway in bacteria may represent a novel approach to control biofilm formation and spread in medical and industrial settings. [0003] It is precisely because c-di-GMP has such an important role that accurate detection of its changes is of great significance for the study of c-di-GMP-mediated signaling pathw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C09K11/06G01N21/64
CPCC07K14/36C09K11/06G01N21/6486C07K2319/60
Inventor 杨弋赵玉政张晓倩陈政达李写
Owner EAST CHINA UNIV OF SCI & TECH
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