High throughput screening method of mycobacterium tuberculosis alpha-D-glucose-1-thymine phosphate transferase inhibitor

Abstract

The invention discloses a high throughput screening method of a mycobacterium tuberculosis alpha-D-glucose-1-thymine phosphate transferase inhibitor. A method for rapidly and accurately measuring the activity of the mycobacterium tuberculosis alpha-D-glucose-1-thymine phosphate transferase is established by the following steps of: cloning an rmlA gene from a mycobacterium tuberculosis H37Rv strain genome by using the molecular cloning technology; simultaneously creating an Escherichia coli engineering strain which highly expresses alpha-D-glucose-1-thymine phosphate transferase; and purifying the engineering strain to obtain mycobacterium tuberculosis alpha-D-glucose-1-thymine phosphate transferase by using affinity chromatography.. The transferase activity measuring method screens a molecular model of the mycobacterium tuberculosis alpha-D-glucose-1-thymine phosphate transferase inhibitor and is used for the high throughout screening of the mycobacterium tuberculosis alpha-D-glucose-1-thymine phosphate transferase inhibitor in combinational compound libraries, traditional Chinese medicine and natural products.

Description

Technical field: [0001] The invention relates to a method for screening anti-tuberculosis drugs, in particular to a method for high-throughput screening mycobacterium tuberculosis α-D-glucose-1-phosphate thymidine transferase inhibitors. Background technique: [0002] Mycobacterium tuberculosis (Mycobacterium tuberculosis) is the pathogenic bacterium that causes tuberculosis (tuberculosis). According to WHO reports, nearly 1 / 3 of the people in the world have been infected with Mycobacterium tuberculosis, and about 90 million new tuberculosis patients are diagnosed every year. Due to the emergence of multi-drug resistant (MDR) strains and extensive drug-resistant (XDR) strains, none of the existing anti-tuberculosis drugs can effectively cure tuberculosis, resulting in about 2 million deaths per year. It has become one of the leading causes of death from infectious diseases in adults worldwide. [0003] At present, the development of new drugs has changed from the most rando...

AI Technical Summary

Problems solved by technology

[0006] However, the detection method with RmlB enzyme protein as the target has the following disadvantages: the substrate dTDP-glucose (dTDP-Glc) is expensive and unstable in solution, so the screening cost is high; the enzymatic reaction time is long, so it is time-consuming and laborious ; Since there is no commercialized reaction product dTDP-4-keto-6-deoxyglucose standard substance, NaOH can only be added to the reaction system to further convert the reaction product into unstable derivatives, and measure its absorbance value at 318nm, Therefore, the detection results are not accurate enough and the repeatability is low; the enzymatic reaction system is relatively large and cannot be completed in a 96-well plate, so high-throughput screening cannot be performed

Images