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Cholesterol metabolism regulating and controlling medicament screening system and method with hydroxymethyl glutaryl cozymase A reductase as target point

A hydroxymethylglutaryl coenzyme and reductase technology, applied in chemical instruments and methods, biological testing, recombinant DNA technology, etc., can solve the problems of long cycle, unsuitable for large-scale testing, and complicated operation.

Active Publication Date: 2012-09-12
CHOLESGEN (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional method of screening compounds generally has obvious shortcomings such as long period, cumbersome operation, poor specificity and sensitivity, non-standard, not suitable for large-scale detection, etc.

Method used

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  • Cholesterol metabolism regulating and controlling medicament screening system and method with hydroxymethyl glutaryl cozymase A reductase as target point
  • Cholesterol metabolism regulating and controlling medicament screening system and method with hydroxymethyl glutaryl cozymase A reductase as target point
  • Cholesterol metabolism regulating and controlling medicament screening system and method with hydroxymethyl glutaryl cozymase A reductase as target point

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Construction of HMGCR(TM1-8)-EGFP Fusion Expression Vector

[0082] It is known that the amino terminal of HMGCR is an eight-times transmembrane domain, whose function is to regulate the stability of the protein. When the intracellular sterol level is too high, this domain binds to sterol directly or indirectly, causing intracellular ubiquitin-protease Body-mediated degradation of HMGCR. The invention clones the gene fragment encoding the amino-terminal eight transmembrane domain of the hamster HMGCR into the green fluorescent protein expression vector. The expression and degradation of the HMGCR(TM1-8)-EGFP fusion protein in the recombinant vector are regulated by sterol. The construction process of the recombinant vector is as follows:

[0083] Genomic RNA of hamsters was extracted by conventional methods. Using a conventional method, using the genomic RNA as a template, reverse transcription PCR amplifies to obtain the genomic cDNA of the hamster.

[0084] Using ...

Embodiment 2

[0106] Construction of Transient Transfection System and Drug Screening of HMGCR(TM1-8)-EGFP Fusion Protein Expression Vector

[0107] The present invention constructs the HMGCR (TM1-8)-EGFP fusion protein expression vector transient transfection system. The HMGCR(TM1-8)-EGFP fusion protein recombinant vector was transiently transfected into a CHO cell line, and observed by a fluorescence microscope, it was confirmed that the HMGCR(TM1-8)-EGFP fusion protein had a higher expression level in the transient transfection system, The cells were further treated with 25-hydroxycholesterol, and by means of fluorescence microscopy, it was confirmed that the HMGCR(TM1-8)-EGFP fusion protein had good degradation regulation in this system.

[0108] The build process is as follows:

[0109] (1) One day before transfection, CHO cells were treated with 5×10 4 Add a 60-mm cell culture dish, add 4ml of DMEM / F12 complete medium containing 5% fetal calf serum, and culture overnight.

[0110] ...

Embodiment 3

[0118] Construction of stable expression system for HMGCR(TM1-8)-EGFP fusion protein

[0119] The invention constructs a stable expression system of HMGCR (TM1-8)-EGFP fusion protein. The HMGCR (TM1-8)-EGFP fusion protein recombinant vector was transfected into a CHO cell line, and the cells were cultured for 10 days in the culture medium of DMEM / F12 containing 5% fetal calf serum 1mg / ml G418 to obtain a monoclonal cell line; Fluorescence microscope observation confirmed that the HMGCR(TM1-8)-EGFP fusion protein had a high expression level in the monoclonal cell line, further treated the cells with active compounds, and determined that HMGCR(TM1-8) 8) -EGFP fusion protein has good degradation regulation in monoclonal cell lines.

[0120] The build process is as follows:

[0121] (1) One day before transfection, CHO cells were treated with 5×10 4 Add to a 100-mm cell culture dish and add 9 ml of DMEM / F12 complete medium containing 5% fetal bovine serum.

[0122](2) For tran...

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Abstract

The invention discloses a screening system which takes HMG-CoA reductase A as a target spot to regulate cholesterol drug. The screening system adopts a cell system which is formed through connecting N-terminal protein fragment of HMG-CoA reductase A with a detectable signal element and guiding the N-terminal protein fragment into cells, to ensure cells to express fusion protein. The screening system can be used for screening substances which takes HMG-CoA reductase A as the target spot for regulating cholesterin.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a cell target screening compound system. More specifically, the invention relates to a system and a method for establishing a hydroxymethylglutaryl-CoA reductase (HMGCR) cell target rapid screening for regulating cholesterol drugs. Background technique [0002] Cholesterol is not only a key component of biofilms, but also a precursor for the synthesis of bile acids and sterol hormones. However, high levels of cholesterol in the body, such as hypercholesterolemia, can directly lead to atherosclerosis, which is the main cause of stroke and coronary heart disease; Alzheimer's disease, obesity, diabetes, etc. are also closely related to cholesterol metabolism disorders. Due to the pathogenicity of high cholesterol, lowering cholesterol is an important component in the treatment and prevention of cardiovascular and other diseases. [0003] The initial synthesis of intracellular cholesterol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00G01N33/68C12N15/62C12N15/63
Inventor 宋保亮唐静洁
Owner CHOLESGEN (SHANGHAI) CO LTD
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