High-throughput screening method for porcine calcium-sensing receptor (CAR) agonist

A screening method and agonist technology, applied in the field of agricultural, animal husbandry and veterinary applications, can solve problems such as incompetence and immature screening, and achieve the effects of simple operation, good specificity and sensitivity

Inactive Publication Date: 2012-01-18
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

Although these classic methods are straightforward, except that capillary electrophoresis is expected to be developed into a high-t

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  • High-throughput screening method for porcine calcium-sensing receptor (CAR) agonist
  • High-throughput screening method for porcine calcium-sensing receptor (CAR) agonist
  • High-throughput screening method for porcine calcium-sensing receptor (CAR) agonist

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Embodiment Construction

[0050] Step 1: Extract total RNA from pig liver, and amplify the pgCAR gene fragment by RT-PCR;

[0051] Pig liver tissue frozen in liquid nitrogen was ground in a mortar with liquid nitrogen all the time, and total RNA was extracted from the powdered liver tissue. The total RNA extraction kit was purchased from Bioteke, and the method was referred to reagent box instructions. The extracted total RNA was subjected to reverse transcription. The reverse transcription kit was purchased from Applied Biosystems, and the method was referred to the kit instructions to form cDNA. Design two primers according to the sequence of pgCAR: the upstream is 5'-CCC AAG CTT GCC ATG GCC AGC GGG GAA GA-3', and the downstream is 5'-CGC GGA TCC TCA GCT GCA GAT CTC CTG GA-3', which are diluted to 20 μM. Use the reverse-transcribed cDNA as a template to amplify the target gene. The amplification system is operated according to the instructions. The total volume is 25 μl. The PCR system is:

[0052...

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Abstract

The invention relates to a high-throughput screening method for a porcine calcium-sensing receptor (CAR) agonist and belongs to the field of the application of agriculture, animal husbandry and veterinarians. The high-throughput screening method comprises the following steps of: extracting total ribonucleic acid (RNA) from a porcine liver, connecting a pgCAR gene fragment obtained through reverse transcription-polymerase chain reaction (RT-PCR) and an eukaryotic expression plasmid, connecting a (NR1)5-TK promoter whole-gene fragment and a reporter plasmid, cotransfecting mammalian cells by using a recombinant expression plasmid, a recombinant reporter plasmid and an internal reference reporter plasmid so as to form stable cell strains, adding medicines to be screened into the stable cell strains, determining the expression level of a reporter gene, evaluating the bioactivities of the medicines, and selecting the medicine with high bioactivity to obtain the pgCAR agonist. A cell screening model established by the method is easy to operate, has good specificity and sensitivity, and can efficiently, quickly and accurately find agonists of porcine CARs.

Description

technical field [0001] The invention belongs to the field of agricultural, animal husbandry and veterinary applications, and specifically relates to a high-throughput screening method for porcine CAR agonists. Background technique [0002] Nuclear receptors are a class of ligand-dependent transcription factors that initiate a cascade of intracellular signal transduction. Nuclear receptors interact with corresponding ligands and co-regulators to regulate the coordinated expression of genes, thus playing an important role in the growth and development, metabolism, cell differentiation and many physiological processes in the body. The typical mode of regulation of gene expression by nuclear receptors is to bind ligands, causing changes in the receptor structure, allowing the interaction with cofactors, thereby regulating gene expression. [0003] Different from the typical model, CAR can regulate the transcription of genes related to steroid, drug, and xenobiotic metabolism in...

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Application Information

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IPC IPC(8): C12Q1/66C12Q1/02C12N15/12C12N15/79C12N5/10G01N21/64
Inventor 石宝明娄蕾单安山
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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