CN-121975010-A - Nanometer antibody for resisting CD63 protein and application thereof

Abstract

The invention discloses a nanometer antibody for resisting CD63 protein and application thereof. Comprising complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of said CDR1, CDR2 and CDR3 are shown in SEQ ID NOS 2, 3 and 4, respectively. The nano antibody has high affinity and high specificity to a CD63 protein extracellular domain (CD 63-ECD), and KD is 48.13 nM. The nano antibody has high affinity, high specificity, excellent stability and easy production, and is beneficial to the application in disease diagnosis, drug delivery and scientific research.

Inventors

• TANG XIXIANG
• WANG FEIFAN
• LI ZENGPENG
• CHEN SIYU
• CHEN CONG
• CHEN MINGLIANG

Assignees

• 福州大学
• 自然资源部第三海洋研究所

Dates

Publication Date

20260505

Application Date

20260211

Claims (10)

1. 1. A nanobody comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are set forth in SEQ ID NOs 3,4, and 5, respectively; preferably, the nanobody has an amino acid sequence shown as SEQ ID NO. 1.
2. 2. A nucleotide, characterized in that, which encodes the nanobody of claim 1; Preferably, the nucleotide sequence is shown as SEQ ID NO. 2.
3. 3. An expression vector comprising the polynucleotide of claim 2.
4. 4. A host cell comprising the nucleotide according to claim 2 or the expression vector according to claim 3.
5. 5. A method for producing the nanobody of claim 1, comprising culturing the host cell of claim 4 and isolating and purifying the nanobody from the culture.
6. 6. Use of the nanobody of claim 1 for binding CD63 protein.
7. 7. A kit for detecting or binding CD63 protein, comprising the nanobody of claim 1.
8. 8. Use of the nanobody of claim 1 in the manufacture of a product for isolating, detecting or targeting exosomes; Preferably, the product is an immunoaffinity chromatography medium, an ELISA detection reagent, an immunofluorescence detection reagent or an in vitro diagnostic device.
9. 9. A product for isolating, detecting or targeting exosomes comprising the nanobody of claim 1.
10. 10. The product of claim 9, wherein the product is an immunoaffinity chromatography medium, an ELISA detection reagent, an immunofluorescence detection reagent, or an in vitro diagnostic device.

Description

Nanometer antibody for resisting CD63 protein and application thereof Technical Field The invention relates to the field of nanobodies, in particular to a nano antibody for resisting CD63 protein and application thereof. Background Antibodies, also known as immunoglobulins, are key effector molecules of the immune system, synthesized and secreted by B lymphocytes, are capable of recognizing and binding epitope with high specificity, and play an irreplaceable role in disease diagnosis and treatment. Traditional antibodies, such as immunoglobulin G, are tetrameric macromolecules composed of two heavy and two light chains linked by disulfide bonds and have a molecular weight up to about 150 kDa. The complex structure gives the complex structure strong functions and specificity, but also has the inherent disadvantages of complex preparation process, high cost, limited stability and the like. More importantly, due to its large molecular size, conventional antibodies perform poorly in terms of tissue penetration, and it is difficult to effectively reach target areas such as inside solid tumors or across the blood brain barrier, which greatly limits their range of application in vivo diagnosis and therapy. In the 90 s of the 20 th century, researchers found a class of structurally unique antibodies in the blood of camelids that naturally deleted the light chain, consisting of only the heavy chain, and were therefore called heavy chain antibodies. The antigen binding function of such antibodies is responsible for its single variable domain, which is known as VHH or nanobody. Nanobody has a molecular weight of only about 15 kDa, which is one tenth of the size of conventional antibodies, but still has complete antigen binding capacity. Due to its unique single domain structure, nanobody exhibits excellent physicochemical properties including high thermal and chemical stability, ability to remain active under extreme pH and high temperature conditions, excellent solubility and ability to be efficiently expressed in prokaryotic systems (e.g., e.coli), and more importantly, its small molecular weight confers its excellent tissue penetration ability to reach targets that are difficult to reach by conventional antibodies. In addition, the complementarity determining regions of nanobodies, particularly the CDR3 regions, are generally longer and conformationally unique, making them capable of recognizing cryptic epitopes, such as active centers of enzymes or clefts in viral surfaces, inaccessible to conventional antibodies. The advantages enable the nano antibody to have huge application potential in the fields of biosensing, cell imaging, targeted therapy, diagnostic reagent development and the like, and become an emerging research hotspot in the field of antibody engineering. Exosomes are a class of extracellular vesicles between 30 and 200 nanometers in diameter, produced by the intracellular endosomal system and released by fusion of the multivesicular body with the cell membrane. The cell-to-cell communication system is wrapped by a phospholipid bilayer, internally carries and enriches active molecules with biological functions such as various proteins, nucleic acids (such as mRNA and miRNA), lipids and the like, and plays an important role in cell-to-cell communication. In recent years, exosomes have been attracting attention due to their great potential in disease diagnosis, prognosis and treatment. For example, tumor cell-derived exosomes carry specific proteins and nucleic acids associated with their parent cells and can be very valuable biomarkers in liquid biopsies. At the same time, exosomes themselves are also considered very promising drug delivery vehicles due to their natural biocompatibility and targeting. However, the efficient, high purity isolation of exosomes as diagnostic markers or therapeutic carriers is a primary and critical technical bottleneck. Currently, methods for separating exosomes mainly include ultracentrifugation, size exclusion chromatography, polymer precipitation, immunoaffinity capture, and the like. Although the ultracentrifugation method is a gold standard, the process is time-consuming, the equipment is expensive and the integrity of exosomes is possibly damaged, the polymer precipitation method is easy to coprecipitate impurities, and the size exclusion chromatography has the problem of co-elution with particles of similar size. Immunoaffinity capture methods are based on antigen-antibody specific reactions, with the highest specificity theoretically, and their core is to recognize the marker proteins on the surface of exosomes. Among the numerous surface proteins of exosomes, the four transmembrane protein family members CD9, CD63 and CD81 are recognized as the most classical exosome markers. Of these, CD63 is the first four transmembrane protein characterized, is specifically and highly expressed on the exosome membrane, and is an ideal target for identifying and isolating exoso