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Modified luciferase mutant protein, bioluminescent probe, probe group, preparation method and detection method

A luciferase and bioluminescence technology, applied in the field of molecular biology, can solve the problems of inability to real-time and accurate response, limited application of nucleic acid markers, restriction of bioluminescence application, etc., to achieve quantitative detection, high signal-to-noise ratio, etc. Effect

Active Publication Date: 2021-06-25
湖北省中医院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, bioluminescent sensing technology has been successfully applied to the detection of biomarkers such as proteins and cells, but its application in the detection of nucleic acid markers such as microRNAs is still very limited.
The main reason for the above problems is that luciferase, which produces bioluminescence, cannot directly respond to changes in nucleic acid content and structure in real time and accurately.
The above problems restrict the application of bioluminescence in the detection of nucleic acid markers

Method used

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  • Modified luciferase mutant protein, bioluminescent probe, probe group, preparation method and detection method
  • Modified luciferase mutant protein, bioluminescent probe, probe group, preparation method and detection method
  • Modified luciferase mutant protein, bioluminescent probe, probe group, preparation method and detection method

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preparation example Construction

[0023] The present invention provides a method for preparing the modified luciferase mutant protein described in the above technical scheme, comprising the following steps: mutating the cysteine ​​at the 166th position of luciferase to serine, and Addition of a linking region containing a cysteine ​​site to the C-terminus of luciferase results in DNA encoding an engineered luciferase mutant protein whose The nucleotide sequence is shown in SEQ ID NO.2; the DNA encoding the transformed luciferase mutant protein is inserted into a plasmid vector to obtain a recombinant plasmid; the obtained recombinant plasmid is transformed into Escherichia coli to obtain a recombinant large intestine Bacillus; culturing the recombinant Escherichia coli to obtain the modified luciferase mutant protein.

[0024] In the present invention, the cysteine ​​at the 166th position of luciferase is mutated into serine, and a linking region containing cysteine ​​site is added to the C-terminus of lucifer...

Embodiment 1

[0035] Preparation of modified luciferase mutant protein

[0036] (1) Construction of the transformed luciferase mutant expression vector: respectively amplify the N-terminal and C-terminal coding sequences of luciferase by PCR, and insert cysteine ​​at the luciferase 166 site Acid codons were mutated to serine codons, and a Linker coding sequence containing cysteine ​​was added after the C-terminal coding sequence; PCR-amplified fragments were recovered by gel electrophoresis and luciferase was amplified by overlap PCR The complete coding sequence of the mutant, and NdeI and HindIII restriction sites were added at the 5' and 3' ends respectively; after the above fragment was recovered by electrophoresis and digested, it was combined with the pET-25(b+) vector digested with NdeI and HindIII connected, and the correctness of the clone was verified by sequencing; the primers used in PCR are shown in SEQ ID NO.6-SEQ ID NO.8.

[0037] (2) Expression and purification of the modifi...

Embodiment 2

[0039] Construction of Bioluminescent Probes for Detecting Nucleic Acid Molecules

[0040] The structure of the bioluminescent probe for detecting nucleic acid molecules provided by the invention is as follows: figure 1 As shown, its construction method is as follows:

[0041] (1) Coupling of double-stranded DNA:

[0042] A) Functionalization of DNA by SMCC: Incubate 1 mM first-strand single-stranded DNA with amino modification at the 5' end and 1.2 mM second-strand single-stranded DNA with Dabcyl group modification at the 3' end in PBS solution for 1 h , wherein the nucleotide sequence of the first strand is as shown in SEQ ID NO.2, and the nucleotide sequence of the second strand is as shown in SEQ ID NO.3; adding the SMCC that the final concentration is 20mM in the above solution, reverse mixing After homogenization, incubate at 25°C for 2 hours; further add 1 / 10 volume of 3M NaAc (pH=5.3), and add 1 volume of isopropanol, incubate at room temperature for 30 minutes; cent...

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Abstract

The invention relates to a modified luciferase mutant protein, a bioluminescent probe, a probe group, a preparation method and a detection method, and belongs to the technical field of molecular biology. The amino acid sequence of the modified luciferase mutant protein provided by the invention is as shown in SEQ ID NO. 1. The modified luciferase mutant protein provided by the invention is obtained by mutating cysteine at the 166 site of luciferase protein into serine and adding cysteine at the C-terminal of luciferase, and controllable and equal-proportion covalent coupling of the luciferase protein and DNA molecules is realized by modifying the luciferase protein. Therefore, the high-signal-to-noise-ratio bioluminescence probe capable of responding to nucleic acid is constructed, accurate sensing of nucleic acid molecules is realized, and the problem that a bioluminescence sensing technology is difficult to apply to the nucleic acid molecules is solved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a modified luciferase mutant protein, a bioluminescence probe, a probe group, a preparation method and a detection method. Background technique [0002] MicroRNAs (miRNAs) are a class of small RNA molecules with a length ranging from 22 to 25 nt. They not only participate in important biological processes such as cell differentiation, but also are key marker molecules for major diseases such as tumors. At present, miRNAs detection methods based on sensing principles such as electrochemistry, fluorescence, and chemiluminescence have been established. However, the existing methods still have the problems of cumbersome operation, susceptibility to environmental factors, and high cost of detection equipment. [0003] Compared with traditional detection technologies, bioluminescent sensing has the advantages of high signal-to-noise ratio, low phototoxicity, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N15/11C12N1/21C12Q1/6844C12Q1/66C12R1/19
CPCC12N9/0069C12N15/70C12Q1/6844C12Q1/66C12Q2525/207C12Q2537/1373C12Q2563/107C12Q2531/119
Inventor 倪维
Owner 湖北省中医院
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