P2Y1/U2OS cell strain and preparation method and application thereof

A P2Y1, cell line technology, applied in biochemical equipment and methods, microorganism-based methods, cells modified by introducing foreign genetic material, etc., can solve the problems of low oral bioavailability, bleeding risk, clopidogrel resistance, etc. , to achieve good application prospects, reduce detection costs, and reduce false positives

Inactive Publication Date: 2015-09-02
KUNMING UNIV OF SCI & TECH
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Problems solved by technology

[0003] At present, antiplatelet drugs, such as clopidogrel and prasugrel, have been on the market for many years, are widely used, and have definite curative effects, but there are also problems such as clopidogrel resistance and bleeding risk; in recent years, antiplatelet drugs targeting the P2Y1 receptor The research on platelet compounds is deepening day by day. The nucleotide derivative MRS2179 is a selective antagonist of P2Y1 receptor, which has antithrombotic activity in vivo and in vitro. It is the most commonly used tool drug for studying P2Y1 receptor, but it has oral bioavailability low defect

Method used

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  • P2Y1/U2OS cell strain and preparation method and application thereof
  • P2Y1/U2OS cell strain and preparation method and application thereof
  • P2Y1/U2OS cell strain and preparation method and application thereof

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Embodiment 1

[0020] Construction of GFP fluorescence-labeled human P2Y1 receptor eukaryotic expression vector P2Y1 / pCMV6, establishment of P2Y1 / U2OS cell line, and detection of P2Y1 receptor agonists by changes in the average number of fluorescent spots in P2Y1 / U2OS cells.

[0021] 1. GFP fluorescently labeled human P2Y1 receptor (such as figure 1 shown) the construction of eukaryotic expression vector P2Y1 / pCMV6 (such as figure 2 shown):

[0022] (1) Amplification of human P2Y1 cDNA (purchased from OriGene): using human P2Y1 cDNA as a template, PCR was used to amplify the full-length human P2Y1 cDNA coding region, with a total length of 1119 bp;

[0023] Its upstream primer: 5'-gaggcgatcgccATGACCGAGGTGCTGTGGCCGGC-3';

[0024] Downstream primer: 5'-gcgacgcgtCAGGCTTGTATCTCCATTCTGCTTG-3' (primers were synthesized by Shuoyang Biotechnology Co., Ltd.).

[0025] (2) Recover and purify the PCR product, connect it to the pMD18-T Vector to obtain the recombinant plasmid P2Y1 / pMD18-T, and use r...

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Abstract

The invention discloses a P2Y1/U2OS cell strain and a preparation method and application thereof and belongs to the technical field of high throughput screening and detection of drugs. The P2Y1/U2OS cell strain is prepared by the following steps: firstly establishing a human P2Y1 receptor fluorescin eukaryotic expression vector P2Y1/pCMV6 and then transfecting a U2SO cell according to a Lipofectamine2000 operating manual; and screening the G418 (400 mu g/ml) in a pressurized manner for 2 weeks to obtain the P2Y1/U2OS cell strain. The P2Y1/U2OS cell strain disclosed by the invention and used for determining the biological activity of a P2Y1 receptor stimulant or an antagonist is easy to standardize and good in repeatability, has the characteristics of high specificity and sensitivity, low using cost and convenient operation for the P2Y1 receptor, and has a good application prospect in drug screening and efficacy evaluation.

Description

technical field [0001] The invention relates to a P2Y1 / U2OS cell line and its preparation method and application, belonging to the technical field of high-throughput screening and detection of drugs. Background technique [0002] P2Y1 receptors belong to G protein-coupled receptors, which are expressed in human heart, brain, blood vessels, placenta, liver, lung and other tissues. The P2Y1 receptor is coupled to the Gq protein. When the receptor is activated, it can activate phospholipase Cβ (PLCβ) and protein kinase C (PKC) to promote intracellular calcium mobilization. Recent studies have shown that P2Y1 receptors play an important role in platelet activation, vasodilation and atherosclerosis. For example: after ADP binds to the P2Y1 receptor, it will trigger the Gq pathway, causing the increase of the cytoplasmic calcium ion level, thereby causing rapid and reversible aggregation of platelets. In endothelial cells, activation of the P2Y1 receptor can lead to coronary vas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/02C12R1/91
Inventor 季秀玲李明瑞魏云林张宽仁林连兵张琦
Owner KUNMING UNIV OF SCI & TECH
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