A nanobody targeting glucose regulatory protein 78 and its application

A technology for regulating proteins and nanobodies, applied in the field of immunology or molecular biology, can solve problems such as the absence of nanobodies, achieve good binding activity, low preparation cost, and good tissue penetration

Active Publication Date: 2022-06-21
SHENZHEN PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there have been no relevant reports on nanobodies with good binding and high affinity to GRP78 protein.

Method used

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  • A nanobody targeting glucose regulatory protein 78 and its application
  • A nanobody targeting glucose regulatory protein 78 and its application
  • A nanobody targeting glucose regulatory protein 78 and its application

Examples

Experimental program
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Embodiment Construction

[0026] The preferred embodiments of the present invention will be further described in detail below.

[0027] 1. In vitro detection of GRP78 membrane expression

[0028] Breast cancer cells 4T-1 were seeded on high-content 96-well microplates at the number of 10,000-20,000 / well; after the cells adhered, they were washed 3 times with PBS, fixed with 0.25% paraformaldehyde for 10 min; blocked with 5% BSA for 1 h ; Dilute anti-GRP78 antibody (1:200) with PBST, incubate for 1 h at room temperature; wash 3 times with PBST, dilute fluorescent secondary antibody (1:1000) with PBST, incubate for 1 h at room temperature; wash 3 times with PBST, with DAPI-containing Mount the slides with a mounting medium, and detect the membrane expression of GRP78 by a high-content cell imager.

[0029] 2. In vivo detection of GRP78 membrane expression

[0030] Construct breast cancer 4T1 and gastric cancer MKN45 tumor models, when the tumor volume reaches 300 mm 3 The tumor tissue was collected a...

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Abstract

The present invention provides a nanobody targeting glucose-regulated protein 78 and its application. The complementarity determining region of the amino acid sequence of the nanobody targeting glucose-regulated protein 78 is CDR1 as shown in SEQ ID NO: 2, such as CDR2 shown in SEQ ID NO:4, CDR3 shown in SEQ ID NO:6; or CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:13 CDR3 indicated. The nanobody targeting glucose regulatory protein 78 of the present invention has an affinity with glucose regulatory protein 78 at the nanomolar level, has relatively high affinity, and has specific recognition ability for the GRP78 protein on the cell membrane surface.

Description

technical field [0001] The invention belongs to the technical field of immunology or molecular biology, and in particular relates to a nanobody targeting glucose regulatory protein 78 and its application. Background technique [0002] Glucose regulated protein 78 (glucose regulated protein 78 kDa, GRP78) is a protein with a molecular weight of 78 kDa discovered by IraPastan et al. in 1977 in chicken embryo fibroblasts cultured without glucose. (binding immunoglobulin protein, BIP) is exactly the same, so it is also called immunoglobulin heavy chain binding protein (BiP) is an important endoplasmic reticulum molecular chaperone. , metastasis, and angiogenesis are critical. The full-length GRP78 protein consists of two functional domains, including a nucleotide-binding domain (NBD), which can bind ATP, and a substrate-binding domain (SBD), which can bind polypeptides / proteins . GRP78 is widely present in the cytoplasm, but under stress conditions such as hypoxia, glucose de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70C12N1/21A61K39/395A61P35/00C12R1/19
CPCC07K16/18C12N15/70G01N33/57492A61P35/00C07K2317/569C07K2317/565C07K2317/567C07K2317/92A61K2039/505
Inventor 徐承超李志杰王惠芳王继刚卓晓凤付春进袁吉民
Owner SHENZHEN PEOPLES HOSPITAL
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