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CN-121975916-A - In-situ detection system for detecting EZH2 mRNA, and preparation method and application thereof

CN121975916ACN 121975916 ACN121975916 ACN 121975916ACN-121975916-A

Abstract

The invention discloses an in-situ detection system for detecting EZH2 mRNA, a preparation method and application thereof. The in-situ detection system comprises a molecular probe compound and a signal amplification compound, wherein the molecular probe compound is formed by constructing dCS 9-mSA fusion protein and sgRNA of specific targeting EZH2 mRNA, the signal amplification compound is a metal organic framework nano material encapsulated with fluorescent molecules, biotin is modified on the metal organic framework nano material, and the molecular probe compound is specifically combined with biotin on the signal amplification compound through Avidin in the dCS 9-mSA fusion protein to realize in-situ marking and signal amplification of the EZH2 mRNA. The in-situ detection system for detecting the EZH2 mRNA provided by the invention solves the contradiction that the specificity and the sensitivity are difficult to combine in the traditional technology, has the advantages of high sensitivity, strong specificity, good fluorescence quenching resistance, mild operation conditions and the like, can realize in-situ visual detection of the tissue slice level EZH2 mRNA, and is especially suitable for auxiliary diagnosis and prognosis evaluation of retinoblastoma.

Inventors

  • JIA RENBING
  • AN YU
  • WEN XUYANG
  • FAN JIAYAN
  • Zhuang ai
  • YANG JIE
  • FAN XIANQUN

Assignees

  • 上海交通大学医学院附属第九人民医院

Dates

Publication Date
20260505
Application Date
20260211

Claims (9)

  1. 1. An in situ detection system for detecting EZH2 mRNA, comprising a molecular probe complex and a signal amplification complex; The molecular probe complex is constructed by dCAS9-mSA fusion protein and sgRNA of specific targeting EZH2 mRNA; The signal amplification compound is a metal organic frame nano material encapsulated with fluorescent molecules, and biotin is modified on the metal organic frame nano material; The molecular probe complex realizes in-situ labeling and signal amplification of the EZH2 mRNA through specific combination of Avidin in dCAS9-mSA fusion protein and biotin on the signal amplification complex.
  2. 2. The in situ detection system for detecting EZH2 mRNA according to claim 1, wherein the amino acid sequence of dCas9-mSA fusion protein comprises a linker peptide linked between the C-terminus of dCas9 protein and the N-terminus of Avidin protein.
  3. 3. The in situ detection system for detecting EZH2 mRNA according to claim 1, wherein the metal-organic framework nanomaterial is assembled from metal ions coordinated with organic ligands; Wherein the metal ion is copper ion, and the organic ligand is pyridine.
  4. 4. The in situ detection system for detecting EZH2 mRNA according to claim 3, wherein the fluorescent molecule is rhodamine B, which is encapsulated inside the pores of the metal organic framework nanomaterial.
  5. 5. The in situ detection system for detecting EZH2 mRNA according to any one of claims 1 to 4, wherein the modification of biotin on the metal organic framework nanomaterial specifically means that the metal organic framework nanomaterial is modified with biotin on the surface by reaction with sulfhydryl polyethylene glycol biotin.
  6. 6. A method of preparing an in situ diagnostics system as claimed in any one of claims 1 to 5, comprising the steps of: S1, preparing dCS 9-mSA fusion protein, namely constructing recombinant plasmid for expressing dCS 9-mSA fusion protein, transforming host bacteria, and purifying after induced expression to obtain dCS 9-mSA fusion protein; S2, preparing sgRNA, namely designing and synthesizing specific sgRNA according to the sequence of the EZH2 mRNA; s3, preparing a molecular probe complex, namely incubating the dCS 9-mSA fusion protein obtained in the step S1 and the sgRNA obtained in the step S2 in a reaction buffer solution, and assembling to form the molecular probe complex; S4, preparing a signal amplification compound: S41, synthesizing a MOF substrate, namely dissolving a copper ion source and pyridine in an organic solvent, heating to carry out coordination reaction, and synthesizing a metal organic framework nano material; S42 Biotin modification, namely performing surface amination modification on the metal-organic framework nanomaterial obtained in the step S41, activating by using a bifunctional crosslinking agent, and then reacting with SH-PEG-Biotin to prepare a biotinylated metal-organic framework nanomaterial; S43, fluorescent molecule encapsulation, namely immersing the biotinylated metal organic framework nano material in rhodamine B solution, encapsulating the rhodamine B inside the material through physical adsorption, and washing and drying to obtain the signal amplification compound.
  7. 7. The method according to claim 6, wherein in step S41, the organic solvent is chloroform, the molar ratio of copper ions to pyridine is 1:2, the heating reaction temperature is 60 ℃ and the time is 90 minutes.
  8. 8. The method of claim 6, wherein in step S42, the molar ratio of the metal-organic framework nanomaterial to SH-PEG-Biotin is 1:3.
  9. 9. A method for in situ detection of EZH2 mRNA in an isolated sample using the detection system of any of claims 1-5, comprising the steps of: (1) Sample pretreatment, namely fixing, dewaxing and permeabilizing a sample containing cells or tissues to be tested; (2) Probe binding, namely dripping the molecular probe complex into the treated sample, incubating at 37 ℃, and then washing to remove unbound probes; (3) Signal amplification, namely dripping the signal amplification complex into the sample treated in the step (2), incubating at 37 ℃, anchoring a fluorescent molecule to a target point by utilizing the specific combination of the Avidin and the biotin, and then washing; (4) And (3) performing fluorescence imaging, namely performing cell nucleus counterstaining on the sample, observing a fluorescence signal of rhodamine B under a fluorescence microscope, and realizing in-situ detection of the EZH2 mRNA according to the fluorescence signal.

Description

In-situ detection system for detecting EZH2 mRNA, and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to an in-situ detection system for detecting EZH2 mRNA, a preparation method and application thereof. Background Retinoblastoma (Retinoblastoma, RB) is the most common primary intraocular malignancy in infants, with higher disability and mortality. Early accurate diagnosis and prognosis evaluation are important for saving the eyesight and life of children. In recent years, the epigenetic marker EZH2 (Enhancer of zeste homolog 2) has been of great interest because of its critical role in the development of RB. EZH2 as a core catalytic subunit of the multi-comb inhibition complex 2 (PRC 2) leads to oncogene silencing by mediating H3K27me3, whose overexpression is closely related to the high invasiveness and poor prognosis of tumors. Therefore, the realization of in-situ detection of the expression level of EZH2, in particular the mRNA transcription level thereof, has important significance for molecular pathological diagnosis of RB. Currently, detection techniques for EZH2 mainly have the following limitations: Protein level detection (IHC/Western blot) although Immunohistochemistry (IHC) is a gold standard for pathological diagnosis, protein expression is often interfered by post-translational modification, and the specificity of antibodies is greatly different, so that the real-time transcription state of genes is difficult to accurately reflect. Traditional mRNA in situ hybridization (FISH/ISH) can detect mRNA, but needs severe conditions such as high-temperature denaturation, strong acid/strong alkali treatment and the like, so that the method is complex in operation and easy to destroy morphological structures of cells and tissues. In addition, conventional fluorescent probes are prone to photobleaching, and the number of fluorescent molecules carried by a single probe is limited, so that the detection sensitivity is low, and mRNA signals with low abundance are difficult to capture. The conventional CRISPR/dCAS9 imaging technology is that RNA imaging by using inactivated Cas9 (dCAS 9) coupled fluorescent protein or fluorescent dye improves specificity without denaturation, but the early technology is usually one-to-one or a few fluorescent molecular markers, has weak signal intensity, usually requires an expensive confocal microscope to observe, and is difficult to realize clear visualization under a common fluorescent microscope of a conventional clinical pathology department. Disclosure of Invention The invention aims to provide an in-situ detection system for detecting EZH2 mRNA, a preparation method and application thereof, solves the contradiction that the specificity and the sensitivity are difficult to be compatible in the traditional technology, and has the advantages of high sensitivity, strong specificity, good fluorescence quenching resistance, mild operation condition and the like. The first aspect of the invention provides an in situ detection system for detecting EZH2 mRNA, comprising a molecular probe complex and a signal amplification complex; The molecular probe complex is constructed by dCAS9-mSA fusion protein and sgRNA of specific targeting EZH2 mRNA; The signal amplification compound is a metal organic frame nano material encapsulated with fluorescent molecules, and biotin is modified on the metal organic frame nano material; The molecular probe complex realizes in-situ labeling and signal amplification of the EZH2 mRNA through specific combination of Avidin in dCAS9-mSA fusion protein and biotin on the signal amplification complex. In one embodiment of the invention, the amino acid sequence of the dCAS9-mSA fusion protein comprises a linker peptide which is linked between the C-terminus of dCAS9 protein and the N-terminus of Avidin protein. In one embodiment of the present invention, the metal-organic framework nanomaterial is assembled by coordination of metal ions and organic ligands; Wherein the metal ion is copper ion, and the organic ligand is pyridine. In one embodiment of the present invention, the fluorescent molecule is rhodamine B, and the fluorescent molecule is encapsulated inside the pores of the metal organic framework nanomaterial. In one embodiment of the invention, the metal organic frame nano material is modified with biotin, specifically, the metal organic frame nano material is modified with biotin on the surface by reacting with sulfhydryl polyethylene glycol biotin. The second aspect of the present invention provides a method for preparing the in situ detection system, comprising the steps of: S1, preparing dCS 9-mSA fusion protein, namely constructing recombinant plasmid for expressing dCS 9-mSA fusion protein, transforming host bacteria, and purifying after induced expression to obtain dCS 9-mSA fusion protein; S2, preparing sgRNA, namely designing and sy