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Preparation method for monoclonal antibody library of tumor mucoprotein glycopeptide epitope

A monoclonal antibody, antigen-determined technology, applied in the fields of genetic engineering and immunology

Inactive Publication Date: 2014-04-16
JIANGSU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]Aiming at the deficiencies of the above-mentioned prior art, the problem to be solved by the present invention is: 1) How can the complex glycopeptide sequence of mucin be systematically studied? Omics research, 2) How to prepare a complete antibody library to recognize cancerous mucin glycopeptides

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  • Preparation method for monoclonal antibody library of tumor mucoprotein glycopeptide epitope
  • Preparation method for monoclonal antibody library of tumor mucoprotein glycopeptide epitope
  • Preparation method for monoclonal antibody library of tumor mucoprotein glycopeptide epitope

Examples

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Embodiment 1

[0028] Example 1: Preparation of monoclonal antibody library for tumor mucin MUC1 glycopeptide epitope

[0029] (1) According to the amino acid sequence of MUC1 ( figure 2 ), using the mathematical method of permutation and combination, deduced all possible glycopeptide sequences of MUC1 in tumorigenesis. Each MUC1 tandem repeat consists of 20 amino acids. Each repeat has 5 sites that can be modified by glycosylation. In this way, we can theoretically deduce the structure of possible glycopeptides. There are 5 with 1 sugar, 10 with 2 sugars, 10 with 3 sugars, 5 with 4 sugars, and 1 with 5 sugars. A total of 31 glycopeptides can be recognized by 31 different antibodies.

[0030] (2) Preparation of immunogen: In order to study the mucin on the surface of tumor cells, we designed a unique immunogen. We selected a tumor cell Jurkat expressing Tn antigen (A Mutant Chaperone Converts a Wild-Type Protein into a Tumor-Specific Antigen, Andrea Schietinger , et al. Science 31...

Embodiment 2

[0038] Purification and preparation of monoclonal antibody Clone 16: the hybridoma was inoculated in the peritoneal cavity of nude mice, and the ascites fluid was collected 10 days later, and purified with Protein A sepharose (Sigma, 2 ml) affinity chromatography column. The purified antibody was verified by SDS-PAGE to have no other protein bands except IgG heavy chain and light chain.

[0039] For the detection of the specific affinity of monoclonal antibody Clone 16 to glycopeptides: 2 μg / ml glycopeptides (in image 3 Glycopeptide 2u (represented) was conjugated with biotin and bound to a 96-well plate coated with streptavidin (1.5 μg / ml). Serially diluted Clone 16 antibody protein (protein concentration as Figure 6 shown) after binding to the glycopeptide, was detected with a goat anti-mouse secondary antibody. Secondary antibody with horseradish peroxidase was detected with DAB kit. The results showed that the antibody (Clone 16) had a high specific affinity for glyco...

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Abstract

The invention relates to a preparation method for a monoclonal antibody library of tumor mucoprotein glycopeptide epitope. The method comprises the following steps of: extrapolating all possibly occurring tumor glycopeptide sequences according to an amino acid sequence of mucoprotein; then transfecting tumor cells by using mucoprotein genes to obtain immunogens for immuning a mice to prepare monoclonal antibodies; synthesizing all extrapolated glycopeptides by using a chemical synthesis method and labeling with biotins respectively to obtain the biotin-labeled glycopeptides for screening the monoclonal antibodies; and finally, immuning the mice by using the immunogens to prepare hybridoma, screening the hybridoma by using the biotin-labeled glycopeptides, and sorting out the monoclonal antibody aiming at each specific glycopeptide structure. In the invention, an immune glycomic method is used for the first time to extrapolate all possibly occurring epitopes of highly glycosylated tandem repeat polypeptide segments during cancerization. The glycopeptide antigens are obtained by using the chemical synthesis method; and then the synthesized glycopeptides are used for tagging the monoclonal antibodies.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and immunology, and in particular relates to a method for preparing a monoclonal antibody library of a tumor mucin glycopeptide epitope. Background technique [0002] Mucin (MUCIN) is a type 1 glycoprotein expressed by epithelial cells, highly expressed on the cell surface, and highly secreted. The mucin family has nineteen members: MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, and MUC20. Their function is to protect the epithelial cells of the digestive tract, respiratory tract, and reproductive tract, and to regulate cell communication and signaling of epithelial cells. [0003] The structure of mucin consists of intracellular domains, transmembrane domains, and extracellular domains. The extracellular domain can be hydrolyzed by proteases and secreted into the blood circulation. The extracellular region consists of highly glycos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C40B40/10C07K16/18
Inventor 周大鹏卡特亚.迈克尔努哈得.依布拉海穆许化溪依戈尔.阿尔梅达李云森
Owner JIANGSU UNIV
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