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A kind of monoclonal antibody of the catalytic subunit of dna-dependent protein kinase and its application

A monoclonal antibody and protein kinase technology, applied in anti-enzyme immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of limited practical application and large side effects of inhibitors

Active Publication Date: 2019-12-10
CHENGDU ZEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a variety of inhibitors have been used to inhibit the function of DNA-PKcs, the side effects of these inhibitors limit their practical application

Method used

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  • A kind of monoclonal antibody of the catalytic subunit of dna-dependent protein kinase and its application
  • A kind of monoclonal antibody of the catalytic subunit of dna-dependent protein kinase and its application
  • A kind of monoclonal antibody of the catalytic subunit of dna-dependent protein kinase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Obtaining the hybridoma cell line 8D3 and the monoclonal antibody it produces.

[0052] 1. Antigen Preparation

[0053] (1) Obtain the target gene

[0054] Trizol Reagent reagent was used to extract the total RNA of HeLa cells, the total RNA was reverse transcribed into cDNA, and the DNA-PKcs gene fragment was amplified by PCR using cDNA as a template.

[0055] (2) Construction of recombinant expression vector

[0056] The PCR product obtained in step (1) was digested and recovered, and ligated into the expression vector pET41a under the action of T4 DNA ligase to construct the recombinant plasmid pET41a-DNA-PKcs. The vector is used to transform expression bacteria after enzyme digestion and sequencing identification.

[0057] (3) Obtain expression strains containing recombinant expression plasmids

[0058] Transform Escherichia coli Rosetta (DE3) competent cells with the recombinant plasmid obtained in step (2), screen with LB solid medium containing k...

Embodiment 2

[0076] Example 2: Identification and Application of Monoclonal Antibody

[0077] 1. Western blot detection and identification of mouse anti-DNA-PKcs monoclonal antibody (anti-DNA-PKcs)

[0078] Extract the cell lysates of various tumor cell lines, adjust the total protein concentration to 2mg / ml, load the sample into 6% SDS-PAGE gel holes, 20ug / well, transfer the protein in the gel to PVDF membrane after electrophoresis , after blocking with 5% skimmed milk powder, immunostaining was carried out with the anti-DNA-PKcs antibody prepared in Example 1: adding the anti-DNA-PKcs prepared and purified in step (3) of Example 1, the working concentration was 1:1000, Incubate at 37°C for 1 hour, then add HRP-labeled goat anti-mouse antibody at a working concentration of 1:20000, react at 37°C for 1 hour, and after developing, it can be seen that each lysate has a 450KD band (see figure 2 ), which is consistent with the literature reports, proving that this antibody is a specific anti...

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Abstract

The invention relates to a monoclonal antibody of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), a hybridoma cell strain secreting the monoclonal antibody and application of the monoclonal antibody.

Description

technical field [0001] The present invention relates to a monoclonal antibody of a catalytic subunit of DNA-dependent protein kinase (catalytic sunbunit of the DNA-dependent protein kinase, DNA-PKcs), a hybridoma cell line secreting the monoclonal antibody and the application of the antibody. Background technique [0002] Ionizing radiation and other factors can cause DNA double-strand breaks in cells. Among various forms of DNA damage, double-strand breaks (double strand breaks, DSBs) are the most serious biological consequences, the most complicated cellular responses, and cancer. The type of DNA damage most closely associated with the problem. In mammalian cells, there are two main pathways for DSB repair, including non-homologous end joining (NHEJ) and homologous recombination (HR). DNA-dependent protein kinase (DNA-PK) is a key protein in the non-homologous end joining pathway, and it is a serine / threonine kinase. The DNA-PK complex can recognize and bind to the ends ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/574G01N33/68
CPCC07K16/40
Inventor 聂子梅邱俊康张泰川蒋洪娇
Owner CHENGDU ZEN BIOSCI
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