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PGC promoter of porcine small intestinal epithelial cell expression and application thereof

An epithelial cell and promoter technology, applied in the field of animal genetic engineering

Active Publication Date: 2019-07-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • PGC promoter of porcine small intestinal epithelial cell expression and application thereof
  • PGC promoter of porcine small intestinal epithelial cell expression and application thereof
  • PGC promoter of porcine small intestinal epithelial cell expression and application thereof

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Embodiment 1

[0026] The acquisition and application of embodiment 1PGC promoter

[0027] Using genetic engineering technology, look up the PGC gene sequence in the Ensembl database, and predict it through Promoter2.0Prediction Server, and get a reliable PGC promoter region. Primers for the PGC promoter were designed using primer premier 5.0, PCR was performed using the Chinese experimental miniature pig genome as a template, and the amplified fragment was ligated with the pGL3-Basic vector. Through sequencing verification, the recombination of the PGC promoter-Luc expression vector was realized. Then the recombinant plasmid was transfected into IPEC-J2 cells, and the luciferase activity was detected. Studies have found that the PGC promoter can achieve the effect of starting Luc (luciferase), see the schematic diagram figure 1 . Methods as below:

[0028] 1. Prediction of PGC reliable promoter region

[0029] The PGC gene sequence was searched through the Ensembl database, and the 900...

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Abstract

The invention provides a PGC promoter of porcine small intestinal epithelial cell expression and application thereof. The PGC promoter has the nucleotide sequence shown in SEQ ID No.1. A PGC reliablepromoter area is predicted through an online website; a primer is synthesized through a chemical synthesizing method; a PGC promoter sequence is inserted in a pGL3-Basic carrier through restriction enzyme cutting sites MluI and NheI to obtain a PGC-dual-luciferase recombinant plasmid; the plasmid is transfected into escherichia coli to be amplified, the plasmid is extracted, and sequencing and verifying are conducted; the plasmid is transfected into a porcine small intestinal epithelial cell, it is found that the porcine PGC promoter can promote the dual-luciferase gene expression in the porcine small intestinal epithelial cell, it is shown that the promoter can regulate and control the expression of a downstream gene in the porcine small intestinal epithelial cell, a stable and efficientsmall intestinal transfection regulation and control element can be easily established, and the PGC promoter has application value in the research on the transgenic animal preparation and the small intestinal disease gene therapy.

Description

technical field [0001] The invention relates to the field of animal genetic engineering, in particular to a PGC promoter expressed by pig small intestinal epithelial cells and its application. Background technique [0002] The promoter is an important cis-acting element in the gene, and it is also an important part of the expression vector of genetic engineering. It is generally located dozens of bases upstream of the transcription initiation site. With the development of genetic engineering, it is often necessary to construct an expression vector that can express heterologous proteins at a high level, and the promoter has a great influence on the expression level of foreign genes, and is an important element in genetic engineering. Therefore, choosing an appropriate promoter plays a crucial role in research. [0003] The small intestine is not only the main place for digestion and absorption, but also an important immune barrier, with a high incidence of enteritis and inte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/85C12Q1/6897
CPCC12N9/6481C12N15/85C12N2800/107C12Q1/6897
Inventor 张然白兰
Owner CHINA AGRI UNIV
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