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CN-116660535-B - Probe for detecting PRMT5, kit and application

CN116660535BCN 116660535 BCN116660535 BCN 116660535BCN-116660535-B

Abstract

The invention provides a probe, a kit and an application for detecting PRMT5, which belong to the technical field of biological medicines, wherein two antibody-DNA conjugates, namely DNA1-Ab1 and DNA2-Ab2, are firstly constructed, and are used as probes for detecting PRMT5, the two antibody-DNA conjugates are assembled into a homogeneous immunoassay kit for detecting PRMT5, when the homogeneous immunoassay kit is used for detecting PRMT5, DNA connected with the two PRMT5 antibody-DNA conjugates is close to generate an ortho effect and hybridized, and then the concentration of target protein PRMT5 is determined by detecting signals generated by DNA hybridization, and the homogeneous immunoassay kit has high sensitivity and high specificity and has good application in detecting PRMT 5.

Inventors

  • TU ZHIGANG
  • LIU HANQING

Assignees

  • 江苏莱森生物科技研究院有限公司

Dates

Publication Date
20260508
Application Date
20230513

Claims (4)

  1. 1. A homogeneous immunoassay kit for detecting PRMT5, wherein the homogeneous immunoassay kit comprises a probe for detecting PRMT5, DNA3 for marking acridinium ester, and graphene oxide coupled antioxidant (GO-AT); the probes are two antibody-DNA conjugates, namely DNA1-Ab1 and DNA2-Ab2; the nucleotide sequence of the DNA1 is shown in SEQ ID NO. 1: 5 ́-GCTTGGTCCGTCCGGGCGAGCACCCGCGCCCCGGGTTTCAAACGGTAGGCT-SH-3’, The nucleotide sequence of the DNA2 is shown in SEQ ID NO. 2: 5.́ -SH-TCAACGAACCCCCGGGAATCCAATAGCCAGCTCGGGGCCCTCACCGAGGT-3', wherein the nucleotide sequence of the DNA3 for marking the acridine ester is shown in SEQ ID NO. 3: SEQ ID NO.3:5 ́-SA-NH 2 C 6 -GGAATCAAGAGGGCCGGACGTTAATGAG-3’; The acridinium ester comprises N, N '-dimethyl-9, 9' -diacetylene dinitrate, and the antioxidant in the graphene oxide coupling antioxidant comprises glutathione, benzanilide or 2-hydroxybenzylaniline; The concentration of the DNA1-Ab1 and the DNA2-Ab2 in the homogeneous immunoassay kit is 0.1-50 nM, the concentration of the DNA3 marked with acridine ester is 0.01-1.0 mu M, and the concentration of the GO-AT is 15 mu g/mL.
  2. 2. The homogeneous immunoassay kit of claim 1, wherein the concentration of DNA1-Ab1 and DNA2-Ab2 in the homogeneous immunoassay kit is 5 nM and the concentration of DNA3 labeled with acridinium ester is 0.2 μm.
  3. 3. A method for detecting PRMT5, characterized in that the homogeneous immunoassay kit for detecting PRMT5 according to any one of claims 1-2 is used for non-therapeutic and diagnostic in vitro detection.
  4. 4. A method of detecting PRMT5 according to claim 3, wherein said detecting process is: the PRMT5 antibody on the probe for detecting PRMT5 specifically binds to the target protein PRMT5, then DNA1 and DNA2 generate an ortho effect and hybridize with DNA3, and the target protein PRMT5 concentration is determined by detecting a chemiluminescent signal.

Description

Probe for detecting PRMT5, kit and application Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to a probe for detecting PRMT5, a kit and application thereof. Background The chemiluminescent immunoassay technology combines a chemiluminescent assay technology with a high specificity immune reaction, and is a technology for analyzing various antibodies, antigens, haptens, small molecules, medicines and the like. Homogeneous phase chemiluminescence immunoassay technology has become a hot spot for the research of immunoassay technology in recent years due to the technical characteristics of high specificity and high sensitivity of the homogeneous phase chemiluminescence immunoassay technology, which has the advantages of no washing and easy automation. At present, the products in the field of in-vitro diagnosis and detection at home and abroad are mainly heterogeneous chemiluminescence. This method relies on physical separation and typically requires lengthy and cumbersome washing steps. Therefore, the whole analysis process of the heterogeneous chemiluminescence method has the advantages of multiple steps, huge instrument and equipment, high failure rate, long detection time, complex operation and higher cost. Compared with the method, the homogeneous phase chemiluminescence immunoassay does not need separation and cleaning steps, and the chemiluminescence detection is directly carried out under the pure liquid phase condition, so that the method has the advantages of few instrument and equipment modules, low failure rate and simple and rapid operation. The existing mature homogeneous phase chemiluminescence immunoassay technology takes a light excitation chemiluminescence technology as a core, and is mainly monopoly of foreign enterprises Siemens and Perkin Eimer at present. However, the method needs laser excitation, has high equipment requirement, and is difficult to obtain stably due to special marking materials. The arginine methyltransferase (PRMT) family of proteins uses methylated arginine residues as post-translational modifications. PRMT5 is a type II PRMT, PRMT5 being the only two most active PRMT enzymes catalyzing sDMA formation, PRMT5 acting as a transcriptional co-inhibitor by histone methylation and has been shown to be critical for RNA splicing, which acts to methylate Sm protein for proper assembly into small nuclear ribonucleoprotein (SnRNPs). PRMT5 regulates or interferes with the expression of a variety of oncogenic or oncostatic proteins, including TP53, FOXP1, FOXP3, SLC7A11, c-Myc, BCL6, BAX, EGFR, and TDP1 proteins. PRMT5 expression is epigenetically upregulated, and its increased expression is directly associated with poor prognosis for many malignancies, including lung cancer, breast cancer, hepatocellular carcinoma and glioblastoma multiforme, and its association with lymph node metastasis has been demonstrated in lung cancer, breast cancer, gastric cancer, head and neck cancer, colorectal cancer and epithelial ovarian cancer, as well as bladder urothelial cancer. Furthermore, PRMT5 expression has been shown to promote drug resistance. Furthermore, PRMT5, in a variety of hematological tumors, modulates through MYC splicing, increasing expression of mutant FLT3, thereby driving tumor progression. The ortho-induced DNA dynamic assembly immunoassay is an emerging homogeneous assay with DNA-assisted protein detection. The method has the characteristics of simplicity and rapidness, converts the detection of the protein into the detection of the DNA, and combines the DNA signal amplification technology, so that the detection sensitivity is extremely high. There is therefore a need to develop a homogeneous immunoassay kit for detection of PRMT5 based on proximity effects. Disclosure of Invention Aiming at the defects in the prior art, the invention provides a probe, a kit and application for detecting PRMT5, wherein two antibody-DNA conjugates, namely DNA1-Ab1 and DNA2-Ab2, are firstly constructed, the two antibody-DNA conjugates are used as probes for detecting PRMT5, the two antibody-DNA conjugates are assembled into a homogeneous immunoassay kit for detecting PRMT5, when the homogeneous immunoassay kit is used for detecting PRMT5, DNA connected to the two PRMT5 antibody-DNA conjugates is close to generate an ortho effect and hybridized, and then the concentration of target protein PRMT5 is determined by detecting signals generated by DNA hybridization, and the homogeneous immunoassay kit has high sensitivity and high specificity and has good application in detecting PRMT 5. In order to achieve the technical purpose, the invention adopts the following technical means: the invention first provides a probe for detecting PRMT5, which comprises a PRMT5 antibody and DNA connected with the PRMT5 antibody; The DNA comprises a sequence shown as SEQ ID NO.1 or SEQ ID NO. 2: SEQ ID NO.1: 5′-GCTTGGTCCGTCCGGGCGAGCACCCGCGCCCCGGGTTTCAAACGGT