Single-domain heavy chain antibody for anti-prostate specific membrane antigen

A single-domain heavy chain antibody, prostate-specific technology, applied in the direction of tumor-specific antigen, tumor rejection antigen precursor, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the complex preparation process, High production cost and other issues, to achieve the effect of good permeability, low cost and good application prospects

Active Publication Date: 2016-09-28
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with single-domain heavy chain antibodies, these ligands have disadvantages such as relatively high production costs and complicated preparation processes.

Method used

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  • Single-domain heavy chain antibody for anti-prostate specific membrane antigen
  • Single-domain heavy chain antibody for anti-prostate specific membrane antigen
  • Single-domain heavy chain antibody for anti-prostate specific membrane antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Eukaryotic Expression of the Extramembrane Region of Prostate Specific Membrane Antigen

[0028] RNAiso Plus reagent was used to extract total RNA in LNCaP cells with high expression of PSMA, and RT-PCR method was used to obtain a DNA fragment encoding the extracellular region of PSMA, which was inserted into eukaryotic expression using Not I and BamH I restriction enzymes The vector pRAG2a was ligated into a recombinant plasmid by T4 DNA ligase. The recombinant plasmid was heat-shocked and transformed into TOP 10 competent cells and cultured overnight, and the correctly identified clones were sent for sequencing verification. The plasmids of positive clones were extracted, and the DNA plasmids were transfected into HEK-293 cells using liposome Lipofectamine 2000 for culture, and the supernatants were collected at different time points for SDS-PAGE electrophoresis detection. After culturing for a certain period of time, the protein was purified according to the operati...

Embodiment 2

[0030] Panning and Identification of Anti-Prostate-Specific Membrane Antigen Single-Domain Heavy-Chain Antibody

[0031] Using solid-phase panning method to display camel-derived natural antibody phage display library (for reference: "Tu Zhui, Xu Yang, Liu Xia, et al. Construction and identification of camel-derived natural single-domain heavy chain antibody library [J]. China Bioengineering Journal, 2011, 31 (4): 31-36." In the display library constructed in), the single-domain heavy chain antibody against prostate-specific membrane antigen was panned. The expression of the extracellular region of prostate-specific membrane antigen was carried out according to the above-mentioned Example 1. During the first round of panning, the protein of the extracellular region of PSMA synthesized above was diluted to 150 μg / mL with phosphate buffered saline solution (PBS, pH7.4) (The coating concentrations of rounds 2-4 are 100, 50, and 50 μg / mL, respectively), add 100 μL to each well of ...

Embodiment 3

[0040] ELISA and fluorescent immunoassay of PSMA expressing cells

[0041] Gastric cancer cell MKN45 does not express PSMA, while prostate cancer cell LNCaP expresses PSMA. Taking these two types of cells as examples, the anti-prostate specific membrane antigen single-domain heavy chain antibody phage-positive clones obtained by panning in Example 2 were used for cell-level detection. ELISA and fluorescent immunoassay. LNCaP cells (expressing PSMA) and MKN45 cells (not expressing PSMA) were seeded into 96-well plates at 1×10 4 , cultured overnight. After fixing with 4% paraformaldehyde, 100 μL of 3% hydrogen peroxide solution was added dropwise to each well to block endogenous peroxidase activity, and incubated at 37°C for 30 min. Wash the plate three times with TBS, block with 5% BSA-PBS, add 100 μL anti-prostate specific membrane antigen single domain heavy chain antibody phage-positive clone, and incubate at 37°C for 1 hour. Then rinse with PBS, add HRP-anti-M13 antibody...

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Abstract

The invention relates to a single-domain heavy chain antibody aiming at anti-prostate specific membrane antigen, which belongs to the field of gene engineering. The antibody has the protein with the amino acid sequence disclosed as SEQ ID NO:1, and can be used in the fields of immune detection, antigen enrichment purification and the like. The amino acid sequence provided by the invention can be used as a precursor, can be modified by using a random or site-directed mutagenesis technique to obtain a mutant with better properties (affinity, specificity, stability and the like), and is used for developing proteins or polypeptides which are further used in medicine, industry and agriculture.

Description

technical field [0001] The present invention relates to single domain heavy chain antibody technology (also known as nanobody technology), and genetic engineering antibody technology, especially single domain heavy chain antibody or polypeptide for prostate specific membrane antigen. technical background [0002] Single-domain antibodies refer to genetically engineered antibodies composed of common antibody variable regions (VH or VL). Single-domain heavy chain antibody (also known as nanobody, VHH antibody, variable domain of heavy chain of heavy-chain antibody) refers to a genetically engineered antibody composed only of a heavy-chain antibody variable region (Variable region) , wherein the heavy-chain antibody (heavy-chain antibody) is an antibody that naturally lacks light chains in animals such as camels and sharks. Single domain heavy chain antibody is the smallest complete antigen-binding fragment known so far. It has the characteristics of small molecular weight and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C07K14/47C07K1/14C12N15/13G01N33/68B01J20/24
CPCB01J20/24B01J2220/4856C07K14/4748C07K16/3069C07K2317/622G01N33/68G01N2333/47
Inventor 郭燕丽范校周
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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