Oligopeptide ATR001 and monoclonal antibody prepared through oligopeptide and having bias AT1R regulating function and application

A monoclonal antibody and short peptide technology, applied in the direction of antibodies, biochemical equipment and methods, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc.

Active Publication Date: 2019-03-15
WUHAN HUAJIYUAN BIOTECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biased regulation of receptors is currently a hot spot in drug development, but no drug that biasedly regulates AT1R has been successfully developed

Method used

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  • Oligopeptide ATR001 and monoclonal antibody prepared through oligopeptide and having bias AT1R regulating function and application
  • Oligopeptide ATR001 and monoclonal antibody prepared through oligopeptide and having bias AT1R regulating function and application
  • Oligopeptide ATR001 and monoclonal antibody prepared through oligopeptide and having bias AT1R regulating function and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] The preparation of embodiment 1 hybridoma cell CQ8-A2D9

[0071] 1. Obtaining mouse splenocytes

[0072] 1) Immunization antigen acquisition

[0073] A short peptide ATR001 with a purity of ≥85% was synthesized for the sequence AFHYESQ, and then the short peptide ATR001 was coupled with the carrier protein KLH as an immune antigen;

[0074] 2) Animal immunity

[0075] Immunize 4-6 6-week-old Balb / c mice (live mice) with immune antigens, regular cycle: 3 weeks-2 weeks-2 weeks... intensive cycle: 2 weeks-2 weeks-1 week...use Mouse antiserum ELISA titer ≥ 1:80000, the specific steps are as follows:

[0076] (1) For the first immunization, the amount of immune antigen used: 50ug / monkey, plus Freund's complete adjuvant subcutaneous multi-point injection, with an interval of 3 weeks;

[0077] (2) The second immunization on the 21st day, the dosage route is the same as above, plus Freund's incomplete adjuvant, and the interval is 2 weeks;

[0078] (3) The third immunizati...

Embodiment 2

[0115] 1. Hybridoma subcloning and strain establishment

[0116] ① Preparation of mouse splenocytes as feeder cells;

[0117] ② Prepare the hybridoma cell suspension to be cloned and dilute it with HT medium containing 20% ​​serum to 3 different dilutions containing 5, 10 and 20 cells per ml;

[0118] ③ Add 5×10 per ml 4 -1×10 5 The ratio of cells, respectively add peritoneal macrophages to the above-mentioned hybridoma cell suspension;

[0119] ④ Pack each type of hybridoma into a 96-well plate, with a volume of 100ul per well;

[0120] ⑤37℃, 5% CO 2 After 6 days of culture, the antibody can be detected when there are clones visible to the naked eye; observe under an inverted microscope, mark the wells where only a single clone grows, and take the supernatant for antibody detection.

[0121] ⑥The cells in the wells with positive antibody detection were taken for expansion, cultured, frozen, and subcloned 2-3 times to obtain 2-5 monoclonal cell lines. Hybridoma CQ8-A2D9 ...

Embodiment 3

[0132] Example 3 Effect of monoclonal antibody anti-AT1R on phosphorylation of ERK and JNK in CHO cells stably expressing AT1R

[0133] 1. Culture AT1R-CHOK1 cell line (PerkinElmer): DMEM / F12 complete medium containing 10% FBS, 5% CO 2 Cell culture incubator;

[0134] 2. Detection of AngⅡ-induced phosphorylation of ERK and JNK in AT1R-CHOK1

[0135] Inoculate AT1R-CHOK1 in a six-well plate with a density of about 80%, and culture overnight. Then they were starved with HBS for 2h, stimulated by AngⅡconcentration gradient and time gradient, and detected the expression of p-ERK / ERK and p-JNK / JNK by Western Blot.

[0136] 3. Detect the effect of monoclonal antibody anti-AT1R on the phosphorylation of ERK / JNK caused by AT1R activation

[0137] See steps 1 and 2 for cell culture and seeding. After HBS starvation for 2 hours, ARB group and monoclonal antibody group were pre-incubated with losartan and monoclonal antibody anti-AT1R for 1 hour, and then all groups (except the contr...

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Abstract

The invention discloses an oligopeptide ATR001 and a monoclonal antibody prepared through the oligopeptide and having bias AT1R regulating function and application. The oligopeptide ATR001 is characterized in that the amino acid sequence is AFHYESQ, the hybridoma cell is CQ8-A2D9; and the storage number is CCTCC NO: C2017108. According to the oligopeptide ATR001, the monoclonal antibody technologyis carried out to prepare the AT1R monoclonal antibody, and the antibody is capable of regulating the AT1R function in a bias manner and is capable of treating vascular remodeling diseases such as high blood pressure, atherosclerosis and arterial aneurysm.

Description

technical field [0001] The present invention relates to a short peptide and a monoclonal antibody, specifically a short peptide ATR001 and the preparation and application of a monoclonal antibody with biased AT1R function prepared from the short peptide. Background technique [0002] The renin-angiotensin system (RAS) is an important system that regulates the physiological functions of the human body. Renin is a proteolytic enzyme produced and secreted by renal juxtaglomerular cells, which can hydrolyze angiotensinogen (AGT) to generate angiotensin Ⅰ (Ang Ⅰ). Angiotensin-converting enzyme (ACE), which is widely distributed in the heart, blood vessels, kidneys, lungs and other tissues, can degrade Ang I into angiotensin II (Ang II). chymase) to generate Ang II, or be directly degraded by endopeptidase into a series of small molecule active substances such as angiotensin 1-7 (Ang1-7). At the same time, angiotensin-converting enzyme 2 (ACE2) can also act on AngⅡ to generate a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/72C12N5/20C07K16/28A61K39/395A61P9/12A61P9/10A61P35/00
CPCA61K2039/505C07K14/723C07K16/2869A61K39/395A61P9/10A61P9/12A61P35/00C07K7/14C07K14/72C07K16/28
Inventor 陈霄
Owner WUHAN HUAJIYUAN BIOTECH DEV
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