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Drug screening cell model with NLRP3 (NLR family, pyrin domain containing 3) as target spot and application of drug screening cell model

A cell model and drug technology, applied in the field of molecular pharmacology, can solve problems such as the unknown state of knowledge, and achieve the effect of obvious innovation value and application prospect

Inactive Publication Date: 2014-03-26
NANJING UNIV
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Problems solved by technology

Unfortunately, so far, people's understanding of the regulation of NLRP3 gene expression, especially the regulation mechanism that occurs at the post-transcriptional level, is still unknown

Method used

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  • Drug screening cell model with NLRP3 (NLR family, pyrin domain containing 3) as target spot and application of drug screening cell model
  • Drug screening cell model with NLRP3 (NLR family, pyrin domain containing 3) as target spot and application of drug screening cell model
  • Drug screening cell model with NLRP3 (NLR family, pyrin domain containing 3) as target spot and application of drug screening cell model

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Embodiment Construction

[0023] 1 Materials and methods

[0024] 1.1 Experimental method

[0025] Dual-Luciferase Reporter Assay System was purchased from Promega. XbaI, SacI restriction endonuclease, Primer STAR HS DNA Polymerase, T4DNA Ligase were purchased from TaKaRa

[0026] Taq DNA Polymerase was purchased from TIANGEN, and DMEM was purchased from HyClone.

[0027] 1.2 Experimental method

[0028] 1.2.1 Clone build

[0029] (1) First, amplify the target fragment from the genome or from the plasmid by PCR method, and recover the target fragment by electrophoresis and gel.

[0030] (2) Digested target fragment and plasmid vector

[0031] (3) Preparation of top10 competent cells (aseptic operation)

[0032] ①Put 45 μl of top10 overnight bacteria in an LB tube (with 3ml), shake at 37°C for 1 hour, until the OD value is between 0.15-0.25 (do not overshoot).

[0033] ② Divide into 2 sterile EP tubes, centrifuge at 4°C, 3400rpm, 5min.

[0034] ③ Discard the supernatant, add 700 μl 0.1M CaCl2 so...

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Abstract

The invention relates to a drug screening cell model with NLRP3 (NLR family, pyrin domain containing 3) as a target spot and an application of the drug screening cell model. According to the cell model, a mammalian cell is used as a host, a recombinant plasmid containing an NLRP3 3'-UTR (untranslated region) core sequence and a reporter gene is transfected, and the NLRP3 3'-UTR core sequence is one of SEQ ID 1-9. According to the cell model, the minimum 3'-UTR sequence which can substitute for the NLRP3 mRNA (messenger ribonucleic acid) overall length is found with a 3'-UTR fragment reduction method, the sequence is connected with the reporter gene, and then the connected recombinant plasmid is transfected into an HCT116 cell strain to form a stable-expression cell strain, then endogenous metabolite and natural compounds to be screened are added, the expression level of the reporter gene is determined, and meanwhile, positive and negative control groups are established, so that the activity of substances to be screened are evaluated, and a sensitive and effective natural innovative drug screening method applicable to high-flux anti-infective drugs is provided.

Description

technical field [0001] The invention relates to a new method for discovering endogenous stable NLRP3 mRNA metabolites and screening exogenous anti-infection natural medicines by utilizing a screening model established by the 3'-UTR stability of NLRP3 gene, which belongs to the field of molecular pharmacology. Background technique [0002] The NLRP3 inflammasome has the function of regulating the chronic inflammatory response of the body, and its composition includes NLRP3 (NLR family, pyrin domain containing3), apoptosis-associated speck-like protein containing CARD (ASC) and cysteine ​​day Cysteine-requiring aspartate protease-1 (Caspase-1), a cytoplasmic sensor of endogenous or exogenous danger signals, is a molecular platform for activating caspase-1, and regulates interleukin- Maturation and secretion of pro-inflammatory cytokines such as 1β (Interleukin-1β, IL-1β) and IL-18. IL-1β is a classic pro-inflammatory cytokine. Activated IL-1β binds to IL-1 receptor on target ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/68C12Q1/66C12Q1/02
Inventor 殷武周日曹丹
Owner NANJING UNIV
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