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A screening method and application of streptococcus suis c-di-amp synthetase inhibitor

A c-di-amp, Streptococcus suis technology for analysis by chemically reacting materials, chemiluminescence/bioluminescence, etc.

Active Publication Date: 2021-10-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] In addition to the above-mentioned methods, there are no reports of other determination methods

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  • A screening method and application of streptococcus suis c-di-amp synthetase inhibitor
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  • A screening method and application of streptococcus suis c-di-amp synthetase inhibitor

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Embodiment 1

[0035] The screening steps of embodiment 1 Streptococcus suis c-di-AMP synthetase inhibitor IPA-3:

[0036] 1. Construction of DacA(99-283) recombinant expression plasmid and protein purification

[0037] After the prokaryotic expression vector pET28a was digested with NcoI and XhoI, it was recovered by 1% agarose gel electrophoresis and purification, and the primers were designed with oligo7 software (see Table 1), using Streptococcus suis SC19 genome (NZ_CP020863) as a template , amplify the intracellular domain fragment of DacA, use homologous recombinase to ligate the vector and PCR fragment ( figure 1 ), transformed into DH5α-competent Escherichia coli, after several hours, positive clones were identified by colony PCR, and plasmids were extracted by shaking the bacteria and sent to relevant commercial sequencing companies for sequencing.

[0038] Transform the recombinant plasmid with correct sequencing into BL21(DE3) competent Escherichia coli, inoculate the transforma...

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Abstract

The invention belongs to the field of drug target screening, and in particular relates to a screening method for c-di-AMP synthetase inhibitor of Streptococcus suis and application thereof. Based on the chemiluminescence method, the DacA enzyme activity is reflected according to the relative luminescence value in the reaction system. The reaction system is composed of Streptococcus suis SC19 c‑di‑AMP synthetase functional domain DacA (99‑283), ATP, enzyme activity reaction buffer and Reagent. According to the high-throughput screening judgment standard, the Z value of the chemiluminescent method, the ratio of the signal to the background and the average value of the coefficient of variation of the signal values ​​of the negative and positive test groups are calculated, and the present invention meets the high-throughput screening standard. The screening of the drug library of the present invention identifies a compound, namely the inhibitor IPA-3, which has inhibitory effect on c-di-AMP synthetase of Streptococcus suis and Gram-positive bacteria of Streptococcus suis.

Description

technical field [0001] The invention belongs to the technical field of drug target screening, and in particular relates to a screening method for a c-di-AMP synthetase inhibitor of Streptococcus suis and an application thereof. The invention relates to a c-di-AMP synthetase of Streptococcus suis, in particular to a screening method and application of a c-di-AMP synthetase inhibitor. Background technique [0002] There is a second messenger molecule c-di-AMP in Gram-positive bacteria, which is catalyzed by c-di-AMP synthetase (DacA) to synthesize two ATP molecules and generate two PPi molecules at the same time, while Gram Streptococcus-negative bacteria lack DacA homologs and c-di-AMP. c-di-AMP is necessary in a variety of Gram-positive bacteria, and it regulates the physiological and biochemical processes of bacteria such as potassium ion transport, osmotic pressure homeostasis and cell wall balance by combining with effector proteins or ribosomal switches (Fahmi T , Port...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 周锐李昊天李婷婷黎璐黄琦
Owner HUAZHONG AGRI UNIV
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