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CN-122011181-A - NPM1 protein polyclonal antibody and preparation method and application thereof

CN122011181ACN 122011181 ACN122011181 ACN 122011181ACN-122011181-A

Abstract

The invention relates to the technical field of biology, in particular to an NPM1 protein polyclonal antibody, a preparation method and application thereof. The NPM1 protein polyclonal antibody provided by the invention is anti-NPM 1 serum, and the amino acid sequence of the NPM1 is shown as SEQ ID NO. 1. The serum titer of the polyclonal antibody is as high as 1:256000, and the polyclonal antibody can be specifically combined with NPM1 protein in tissues and cells of poultry animals, has the advantages of high sensitivity, wide application range and the like, and lays a foundation for the detection of the NPM1 protein in the later stage and the research of pathological mechanism of virus infection diseases.

Inventors

  • CHEN ZHENG
  • YU JIABANG
  • GUAN SHUWEN
  • GUO ZHENYU
  • Lv Zongyang
  • XIE HUALI
  • WU HUANSHENG

Assignees

  • 江西农业大学

Dates

Publication Date
20260512
Application Date
20260121

Claims (10)

  1. 1. An NPM1 protein polyclonal antibody is anti-NPM 1 serum, and the amino acid sequence of NPM1 is shown as SEQ ID NO. 1.
  2. 2. The NPM1 protein polyclonal antibody of claim 1, wherein the NPM1 is a chicken-derived recombinant protein, and the NPM1 protein polyclonal antibody is a chicken NPM1 protein polyclonal antibody.
  3. 3. A method for preparing an NPM1 protein polyclonal antibody, comprising: providing NPM1 protein with an amino acid sequence shown as SEQ ID NO.1, and preparing antigen; The antigen is immunized on animals for a plurality of times, and the animal immune serum is the anti-NPM 1 serum.
  4. 4. The polyclonal antibody of NPM1 protein according to claim 3, wherein in the step of preparing antigen, the NPM1 protein is mixed with Freund's complete adjuvant in equal volume, and/or The step of immunizing animals with the antigen comprises subcutaneously injecting the antigen into New Zealand white rabbits at multiple times, enhancing immunity once every 10-20 days, collecting blood after 30-40 days, centrifuging to obtain the anti-NPM 1 serum, and/or The nucleotide coding sequence of the NPM1 protein is shown as SEQ ID NO.2, the nucleotide sequence of an upstream primer for amplifying the nucleotide coding sequence is shown as SEQ ID NO.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 4.
  5. 5. Use of the NPM1 protein polyclonal antibody of claim 1 or 2 or the NPM1 protein polyclonal antibody produced by the production method of any one of claims 3 to 4 in the production of an NPM1 protein binding reagent.
  6. 6. Use of the NPM1 protein polyclonal antibody of claim 1 or 2 or the NPM1 protein polyclonal antibody produced by the production method of any one of claims 3 to 4 in the production of an NPM1 gene expression level detection reagent.
  7. 7. The use according to claim 6, wherein the NPM1 gene expression level detection reagent is an enzyme-linked reaction adsorption test detection reagent comprising anti-NPM 1 serum, And coating the anti-NPM 1 serum serving as a capture antibody on the surface of a solid phase carrier, combining an antigen expressed by the NPM1 gene with the capture antibody, and then combining with an enzyme-labeled detection antibody for color development.
  8. 8. The use of the NPM1 protein polyclonal antibody as claimed in claim 1 or 2 or the NPM1 protein polyclonal antibody prepared by the preparation method as claimed in any one of claims 3 to 4 in the preparation of reagents for detecting infection by tambusu virus, The virus infection reagent comprises a tembusu virus infection reagent.
  9. 9. The use according to claim 8, wherein the NPM1 protein detected in the tambusu virus infected sample is more expressed in the cytoplasmic fraction and less expressed in the nuclear fraction.
  10. 10. Use of the NPM1 protein polyclonal antibody of claim 1 or 2 or the NPM1 protein polyclonal antibody produced by the production method of any one of claims 3 to 4 in resolving the mechanism of tambusu virus infection.

Description

NPM1 protein polyclonal antibody and preparation method and application thereof Technical Field The invention relates to the technical field of biology, in particular to an NPM1 protein polyclonal antibody, a preparation method and application thereof. Background NPM1 (Nucleophosmin.sup.1, nucleolin-1) is a multifunctional protein that is widely distributed in the nucleolus and plays an important role in the physiological and pathological processes of cells. NPM1 has multiple key roles in nucleolus function. First, it is involved in regulating ribosomal biogenesis. Nucleoli is the center of ribosomal biogenesis, and NPM1 promotes the correct folding and assembly of ribosomal precursors by binding to rRNA (ribosomal RNA) and ribosomal proteins. It binds to the 45S precursor rRNA, helping to process and mature ribosomal subunits, and also participates in the assembly and transport of ribosomal subunits by modulating proteins in nucleoli. Second, in cellular stress, NPM1 is dynamically repositioned when cells are stimulated by, for example, DNA damage or oxidative stress. It translocates from nucleoli to nuclear cytoplasm, eliminates damaged or misfolded ribosomal proteins, and maintains intracellular homeostasis. Meanwhile, NPM1 interacts with p53 and ARF proteins, regulates cell cycle arrest or apoptosis, responds to stress signals, and prevents abnormal cell proliferation. Furthermore, NPM1 is critical for maintenance of nucleolar structure. The kernel consists of a Fiber Core (FC), a Dense Fibrous Component (DFC) and a particulate component (GC), where NPM1 is primarily located, responsible for maintaining the integrity of the kernel. During nucleolar stress or dysfunction, changes in the distribution and function of NPM1 can affect nucleolar morphology and function, even leading to nucleolar disintegration. In addition, NPM1 has a Nuclear Export Signal (NES) and a Nuclear Localization Signal (NLS) that are capable of dynamic transport between nucleoli and cytoplasm, maintaining functional activity of nucleoli by binding and regulating the entry and exit of other nucleoli proteins, e.g., regulating the entry and exit of p53 protein, affecting its stability and function, and thus regulating cell cycle and apoptosis. Finally, NPM1 plays an important role in DNA damage repair, it binds and stabilizes DNA repair related proteins, maintains genomic stability, and also regulates chromatin structure and gene expression through nucleolar function, ensuring normal DNA replication and transcription activity of cells. In the medical field, the nuclear phosphoprotein encoded by the NPM1 gene plays an important role in the aspects of material metabolism, cell cycle regulation, apoptosis and the like of cells. Mutation or overexpression of the NPM1 gene can lead to uncontrolled cell proliferation and blocked apoptosis, thus triggering tumors. In some types of leukemia, lymphoma, multiple myeloma and other hematological tumors, mutation or overexpression of NPM1 gene can be detected, so NPM1 has become an important diagnostic marker and therapeutic target for hematological tumors. Studies have also demonstrated that the RNA-binding protein NPM1 on the cell surface can be an effective, selective target for the treatment of Acute Myelogenous Leukemia (AML) and solid tumors. In malignant AML cells, NPM1 appears on the cell surface, and the content of leukemia cell surface NPM1 is more than 10-fold higher than that of healthy cells, and the difference between cancerous hematopoietic stem cells and healthy hematopoietic stem cells is more than 100-fold. Therefore, NPM1 plays an important role in physiological and pathological processes of cells and tissues, and how to realize high-sensitivity detection in multiple species is a constraint factor for the rapid development of the technical field. Disclosure of Invention In order to solve the problems, the invention provides an NPM1 protein polyclonal antibody, a preparation method and application thereof, so as to realize high-sensitivity detection of NPM1 proteins in multiple species. In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the invention provides an NPM1 protein polyclonal antibody, which is anti-NPM 1 serum, wherein the amino acid sequence of NPM1 is shown as SEQ ID No. 1. In some embodiments, the NPM1 is a chicken-derived recombinant protein and the NPM1 protein polyclonal antibody is a chicken NPM1 protein polyclonal antibody. Experiments prove that, for example, an enzyme-linked immunosorbent assay takes NPM1 protein as an antigen and takes NPM1 protein polyclonal antibody as a primary antibody, and when the coating concentration of the antigen is 2.5 mug/mL, the serum titer of the NPM1 protein polyclonal antibody is as high as 1:256000. In addition, the Western Blot detection experiment and the indirect immunofluorescence detection experiment show that the expression of the NPM1 protein is m