Novel method for screening angiotensin invertase inhibitor

An angiotensin-converting enzyme technology, which is applied in the field of screening angiotensin-converting enzyme inhibitors, can solve the problems of decreased enzyme activity, high price, and cumbersome steps, and achieves reduced enzyme inactivation, high accuracy, and good repeatability Effect

Inactive Publication Date: 2016-08-17
CHINA PHARM UNIV
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Problems solved by technology

The above two methods need to use a large amount of pure enzymes. The main source of ACE is to buy commercial ACE, and the other is to extract from animal tissues. Commercial pure enzymes are expensive, and the steps of extracting and purifying ACE from tissues are cumbersome and easy to cause Enzyme activity decreased or even inactivated

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  • Novel method for screening angiotensin invertase inhibitor
  • Novel method for screening angiotensin invertase inhibitor
  • Novel method for screening angiotensin invertase inhibitor

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Embodiment

[0037] 1 Experimental materials and instruments

[0038] (1) Cells and plasmids: 293T / 17 cells (ATCC, USA); Competent DH5α cells (Nanjing Arendi, China); ACE plasmid (origene, USA).

[0039] (2) Reagents: DMEM medium (Corning, U.S.); Fetal bovine serum (Gibco, U.S.); Penicillin 10kU / mL and streptomycin 10kU / mL (Invitrogen, U.S.); Opti-MEM medium (Invitrogen, U.S.) ; SuperSignal West Pico chemiluminescence substrate (Thermo, the United States); Chromatographically pure acetonitrile (Tedia, the United States); Milli-Q system preparation (Millipore, the United States); hippuryl-histidyl-leucine (sigma, the United States) ; hippuric acid (sigma, USA); captopril (sigma, USA); propranolol (sigma, USA); ANTI-DDK magnetic beads (origene, USA).

[0040] (3) Transfection reagent:

[0041] Exfect TM Transfection reagent (Vazyme, China); Lipofectamine 2000 (Invitrogen, USA); Lipofectamine 3000 (Invitrogen, USA).

[0042] (4) Three kinds of lysates:

[0043] A: RIPA-NP40 (2mM Tris-HCl...

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Abstract

The invention relates to a novel high-efficiency method for screening an angiotensin invertase inhibitor. According to the method, a FLAG label is utilized to further purify angiotensin invertase overexpressed in mammal cells and immobilize the angiotensin invertase on a magnetic bead, and then the magnetic bead is applied to screening of active components of crude drugs; and high performance liquid chromatography-mass spectrography is employed for tracking assessment of invertase inhibition activity, so the screening method is more sensitive. The method provided by the invention overcomes problems in rapid preparation of pure enzyme; the functions of enzyme are maximally guaranteed through overexpression of target enzyme via eukaryotic cells; and compared with extraction of enzyme from animal tissue, the method is simpler to operate, so inactivation of enzyme caused by complex operation is mitigated and purifying cost for enzyme preparation is substantially reduced.

Description

technical field [0001] The invention relates to a new method for screening angiotensin-converting enzyme inhibitors, which belongs to the field of biotechnology. Background technique [0002] Hypertension is an important disease that threatens human health. Angiotensin converting enzyme (ACE) widely exists in human tissues and plasma. On the other hand, the kinin with vasodilation effect is inactivated and converted into inactive bradykinin, resulting in an increase in blood pressure. ACE inhibitors (ACE inhibitor, ACEI) have been used clinically as first-line drugs for the treatment of hypertension. Existing antihypertensive drugs such as captopril, enalapril, lisinopril, and quinapril can significantly inhibit ACE The activity plays a role in lowering blood pressure, but the synthetic ACEI has adverse reactions such as dry cough, taste disturbance and rash (Duan Lian, World Journal of Integrated Traditional Chinese and Western Medicine, 2014, (11): 1247-251). Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/08
CPCG01N30/02G01N30/08G01N2030/027
Inventor 陈君贾冰洁汤维维李萍
Owner CHINA PHARM UNIV
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