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Epidermal growth factor receptor monoclonal antibody and application thereof

A technology of epidermal growth factor and monoclonal antibody, applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, microorganism, etc., can solve the problem of easily inducing cancer, enhancing cell signal transduction, epidermal growth factor Receptor mass production, etc.

Active Publication Date: 2016-08-31
CHENGDU ZEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, if the gene is mutated, it will lead to a large number of epidermal growth factor receptors, enhance the conduction of cell signals, and easily induce cancer (2)

Method used

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  • Epidermal growth factor receptor monoclonal antibody and application thereof
  • Epidermal growth factor receptor monoclonal antibody and application thereof
  • Epidermal growth factor receptor monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Obtaining of hybridoma cell line 6H11 and the monoclonal antibody it produces

[0050] 1. Antigen preparation

[0051] (1) Obtain the target gene

[0052] Trizol Reagent reagent was used to extract the total RNA of HeLa cells, the total RNA was reverse-transcribed into cDNA, and the EGFR gene was amplified by PCR using the cDNA as a template.

[0053] (2) Construction of recombinant expression vector

[0054] The PCR product obtained in step (1) was double digested and recovered, and ligated into the expression vector pET41a under the action of T4 DNA ligase to construct the recombinant plasmid pET41a-EGFR. The vector is used to transform expression bacteria after enzyme digestion and sequencing identification.

[0055] (3) Obtain expression strains containing recombinant expression plasmids

[0056] Transform Escherichia coli Rosetta (DE3) competent cells with the recombinant plasmid obtained in step (2), screen with LB solid medium containing kanamyc...

Embodiment 2

[0074] Example 2: Identification and Application of Monoclonal Antibody

[0075] 1. mouse anti EGFR Monoclonal antibodies (anti-EGFR) Western blot detection for identification of

[0076]Extract the cell lysates of various tumor cell lines, adjust the total protein concentration to 2mg / ml, load the sample into 8% SDS-PAGE gel holes, 20ug / well, and transfer the proteins in the gel to PVDF membrane after electrophoresis , after blocking with 5% skimmed milk powder, immunostaining was carried out with the anti-EGFR antibody prepared in Example 1: add the anti-EGFR prepared and purified in step (3) of Example 1, the working concentration was 1:1000, and incubated at 37 degrees for 1 hour, then add HRP-labeled goat anti-mouse antibody at a working concentration of 1:20000, react at 37 degrees for 1 hour, and each lysate has a band of 170Kd (see figure 2 ), this antibody is a specific antibody against EGFR; the antibody dilution gradient reaches 1:1000 (antibody working...

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Abstract

The invention relates to an epidermal growth factor receptor monoclonal antibody and a hybridoma cell line secreting the monoclonal antibody. The monoclonal antibody can be used for application in a kit for detection of cancer cells, wherein the cancer cells preferably comprise epidermis tumor, head and neck cancer, breast cancer, renal cancer, pancreatic cancer, non-small cell lung cancer, prostate cancer and colon cancer; and the monoclonal antibody is used for treatment of related diseases.

Description

technical field [0001] The invention relates to a monoclonal antibody of epidermal growth factor receptor (EGFR), a hybridoma cell line secreting the monoclonal antibody and the application of the antibody. Background technique [0002] The activation of proto-oncogenes will lead to gene mutations, which are closely related to the occurrence and development of malignant tumors. The epidermal growth factor receptor (EGFR) is mainly located on the plasma membrane of the cell and belongs to the family of receptor tyrosine kinases. After being activated by ligands such as epidermal growth factor (EGF) and other ligands, it triggers cell The endodomain forms tyrosine kinase activity and further activates downstream cell signaling pathways to complete the transmembrane signaling process (1). The coding gene of epidermal growth factor receptor (EGFR) is a proto-oncogene, which does not express or only expresses in a limited way under normal physiological conditions, and is not car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/30G01N33/577G01N33/574G01N33/68A61K39/395A61P35/00
CPCC07K16/2863G01N33/57484G01N33/577G01N33/68
Inventor 聂子梅邱俊康张泰川蒋洪娇
Owner CHENGDU ZEN BIOSCI
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