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Screening method for dihydrolipoyl succinyltransferase inhibitor

A dihydrolipoic acid succinyl and screening method technology, applied in the field of biochemistry, can solve problems such as lack and difficulty in research and development of DLST inhibitors

Active Publication Date: 2019-04-16
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of an effective DLST inhibitor screening platform, the research and development of DLST inhibitors is still very difficult. Therefore, the construction of an efficient DLST inhibitor screening method is of great significance for the development and functional research of DLST inhibitors.

Method used

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  • Screening method for dihydrolipoyl succinyltransferase inhibitor
  • Screening method for dihydrolipoyl succinyltransferase inhibitor
  • Screening method for dihydrolipoyl succinyltransferase inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: In vitro screening results of mesylconazole on Xoo's total protein

[0081] Take 100mL OD 600 Xoo bacterial solution = 0.6, centrifuged at 7500 rpm for 10 min, discard the supernatant, and then wash 3 times with PBS. Take 10mL of PBS (pH=7.2) to resuspend the cells, place the centrifuge tube on ice for ultrasonic disruption, measure the protein concentration by the bradford method, and then dilute to 1mg / mL. Take 43μL of total protein, add 1μL of mesylconazole with different concentrations to make the final concentration of 50, 100, 250, 500μM respectively, add equal volume of DMSO to the blank group, and incubate at 25℃ for 2h. Then add 5μM probe 1 respectively, and continue to incubate for 2h. Add biotin-N to the above system respectively 3 , Sodium Ascorbate and BTTAA / CuSO 4 The mixed solution undergoes a click reaction. After 2h, add 2×loading buffer to the system, and boil at 95℃ for 10min. Separately take 10 μg samples for SDS-PAGE, then transfer the me...

Embodiment 2

[0082] Example 2: In vitro screening results of mesylconazole on total protein of Citrus canker (Xac)

[0083] Take 100mL OD 600 Xac bacterial solution = 0.6, centrifuged at 7500 rpm for 10 min, discard the supernatant, and then wash 3 times with PBS. Take 10mL of PBS (pH=7.2) to resuspend the cells, place the centrifuge tube on ice for ultrasonic disruption, measure the protein concentration by the bradford method, and then dilute to 1mg / mL. Take 43μL of total protein and add 1μL of mesylconazole with different concentrations to make the final concentration of 10, 25, 50, 100, 250μM respectively. The blank group is added with equal volume of DMSO and incubated at 25℃ for 2h. Then add 50μM probe 2 respectively, and continue to incubate for 2h. Add biotin-N to the above system respectively 3 , Sodium Ascorbate and BTTAA / CuSO 4 The mixed solution undergoes a click chemistry reaction. After 2h, add 2×loading buffer to the system, and boil at 95℃ for 10min. Take 10 μg samples for ...

Embodiment 3

[0084] Example 3: In vitro screening results of mesylconazole for recombinant expression of DLST

[0085] Recombinant plasmid was constructed with pet28a as a vector, introduced into BL21 strain, and then expanded and cultivated. After affinity purification by Ni column, it was digested with thrombin, and finally purified by molecular sieve to obtain recombinant DLST. Take 25ng of recombinant DLST and add biotin-N 3 , Sodium Ascorbate and BTTAA / CuSO 4 The mixed solution undergoes a click chemistry reaction. Then add 2×loading buffer to the system and boil at 95℃ for 10min. Respectively take 12.5ng of recombinant DLST for SDS-PAGE, then transfer the membrane at 200mA for 50min, wash the membrane with PBST and block with 5% skimmed milk powder for 2h. After washing the membrane with PBST, add HRP-labeled streptavidin (HRP-Streptavidin) and incubate for 2h, then add HRP-labeled streptavidin (HRP-Streptavidin) and incubate for 2h, wash the membrane with PBST and add chemiluminescenc...

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Abstract

The invention relates to a screening method for a dihydrolipoyl succinyltransferase inhibitor. Screening of a dihydrolipoyl succinyltransferase inhibitor is carried out under in-vivo and living body conditions. A to-be-screened inhibitor is used for preprocessing total protein or recombinant dihydrolipoyl succinyltransferase or living cells; a probe capable of marking the dihydrolipoyl succinyltransferase is used for processing a reaction system; a click chemical reaction is carried out and coupling of Biotin-N3 is carried out; and then an imaging analysis is carried out by using a streptavidin blot method. The method has the great significance in screening the dihydrolipoyl succinyltransferase inhibitor.

Description

Technical field [0001] The invention relates to the field of biochemistry, and relates to a method for screening dihydrolipoic acid succinyltransferase inhibitors, and specifically, to a method for in vitro and in vivo screening of dihydrolipoic acid succinyltransferase inhibitors . Background technique [0002] Dihydrolipoate succinyltransferase (DLST), as the core component of the α-ketoglutarate dehydrogenase complex, catalyzes the conversion of α-ketoglutarate into succinyl-CoA Plays a key role in the tricarboxylic acid cycle (TCA) [1-3] . Studies have shown that DLST protein is closely related to the occurrence of many diseases, such as Alzheimer's disease [4-6] ,leukemia [7] ,Cardiovascular diseases [8] Therefore, DLST protein is an important disease treatment target, and DLST inhibitors have become potential drugs related to disease treatment. Currently, there are almost no high-efficiency and highly selective DLST inhibitors, which leads to great challenges for the func...

Claims

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Application Information

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IPC IPC(8): G01N21/76A61K31/4245A61P25/28A61P9/00A61P35/02
CPCA61K31/4245A61P9/00A61P25/28A61P35/02G01N21/76
Inventor 杨松陈彪龙青素赵永亮吴元元薛伟
Owner GUIZHOU UNIV
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