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Preparation of monoclonal antibody of OTUD5 and application of OTUD5 in cancer therapy

A monoclonal antibody and breast cancer technology, applied in the field of medicine and biology, can solve problems that other OUT family research does not involve

Active Publication Date: 2017-09-29
PRESEE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although well-studied, studies of other OUT families have not addressed

Method used

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  • Preparation of monoclonal antibody of OTUD5 and application of OTUD5 in cancer therapy
  • Preparation of monoclonal antibody of OTUD5 and application of OTUD5 in cancer therapy
  • Preparation of monoclonal antibody of OTUD5 and application of OTUD5 in cancer therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Screening out OTUD5 in nude mice as a new gene associated with breast cancer metastasis

[0028] 1. In vivo screening model in nude mice

[0029] In this study, we designed an in vivo metastasis model using nude mice to screen deubiquitinating enzymes that can inhibit breast cancer metastasis, and applied it to MDA-MB-231 cells with epithelial-like traits and low metastatic ability. We used A shRNA library targeting 75 deubiquitinating enzymes (the library was purchased through Sigma mission library and Beijing Aurui Dongyuan Biotechnology Co., Ltd.), each deubiquitinating enzyme has 4-6 different shRNAs, of which at least Two have been identified as effective. First, we packaged and produced 371 different shRNA viruses using HEK293T cells, and infected them into a sufficient number of MDA-MB-231 cells in a 12-well plate. After 48 hours, we used Puromycin at a concentration of 2 μg / ml to obtain 371 stable cell lines after 3 days of selection. We then mixed ...

Embodiment 2

[0031] Example 2 Down-regulation of OTUD5 expression inhibits lung metastasis of breast cancer

[0032] In order to verify the role of OTUD5 in tumor metastasis, we used shRNA interference technology (OTUD5 (Locus ID55593) Human shRNA, purchased from Beijing Aurui Dongyuan Biotechnology Co., Ltd.) to screen and obtain OTUD5 knockout MDA-MB-231 cells Department, from figure 2 It can be seen that the mRNA expression of OTUD5 was reduced by 80%, image 3 The amount of protein expression was calculated by graphing software, and the amount of protein expression was reduced by 79%. This shows that the gene knockout cells were constructed successfully.

[0033] After injecting this cell line into the hearts of nude mice, there were 30 mice in each group, and one group was the MDA-MB-231 cell line without knockout. After 8 weeks of reaction, the bone metastasis of the shRNA-introduced cell line was only 10%, while the bone metastasis of the cell line without shRNA reached 66.7%, w...

Embodiment 3

[0034] Preparation of embodiment 3 OTUD 5 monoclonal antibody

[0035] 1. Generation of CHO cells stably expressing OTUD 5 antigen

[0036] According to the NM_001136157 sequence, primers were designed to amplify the full-length human OTUD 5 cDNA, which was connected to the expression vector pcDNA3.1(+) to construct the eukaryotic expression vector pcDNA3.1(+)-OTUD 5.

[0037] (2) Transfection of CHO cells

[0038] Inoculate 3X10 in a 3.5cm tissue culture dish 5 CHO cells, when the cells were cultured to 90%-95% confluence, transfected: 10 μg of plasmid (pcDNA3.1(+)-OTUD 5) and 20 μl of Lipofectamine2000 Reagent (product of Invitrogen Company) were dissolved in 500 μl of serum-free DMEM medium respectively, and static at room temperature Set aside for 5 minutes, mix the above two liquids, and incubate at room temperature for 20 minutes to form the DNA-liposome complex, during which time, replace the serum-containing medium in the petri dish with 3ml of serum-free DMEM medium, ...

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Abstract

The invention discloses preparation of a monoclonal antibody of OTUD5 and an application of the OTUD5 in cancer therapy. Through a lot of experiments and data analysis, the results show that OTUD5 is closely associated with occurrence and transfer expression of a breast cancer, the OTUD5 can be applied to treatment of the breast cancer as a therapeutic target. In addition, through improved monoclonal antibody of the OTUD5, the antibody has relatively good binding characteristics.

Description

technical field [0001] The invention relates to the application of monoclonal antibody of OTUD 5 in cancer treatment, and belongs to the field of medical biotechnology. Background technique [0002] The ubiquitin-proteasome pathway is an important regulatory system for protein degradation in cells. Through polyubiquitination of substrate proteins and degradation by proteasomes, it can affect or regulate a variety of cellular activities, including: gene transcription, cell cycle regulation, immune response, cell receptor function, tumor growth, and inflammatory processes. This pathway is also a tightly regulated reversible process, in which the regulation of deubiquitinating enzymes is an important link. Current studies have confirmed that there are many deubiquitinating enzymes (DUBs) widely in cells, which are mainly divided into five types: the ubiquitin carboxy-terminal hydrolase family and the ubiquitin-specific processing enzyme family. Among them, the OUT (Otubain Pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40G01N33/574G01N33/577A61K39/395A61K31/7105A61K48/00A61P35/00
CPCA61K31/7105A61K2039/505C07K16/40C07K2317/56G01N33/57415G01N33/577
Inventor 王振谢枫
Owner PRESEE BIOTECH CO LTD
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