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Expression vector capable of efficiently expressing cow menin in eukaryotic cells

An expression vector and high-efficiency expression technology, applied in the field of animal genetics and animal molecular cell biology

Inactive Publication Date: 2015-06-10
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the research on MEN1 in China is still discussing the relationship between MEN1 gene polymorphism and tumor development, or the influence of a certain section of MEN1 gene on cells. Specific Mechanisms of Cell Biological Effects

Method used

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  • Expression vector capable of efficiently expressing cow menin in eukaryotic cells
  • Expression vector capable of efficiently expressing cow menin in eukaryotic cells
  • Expression vector capable of efficiently expressing cow menin in eukaryotic cells

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Experimental program
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Effect test

Embodiment 1

[0031] An expression vector capable of efficiently expressing bovine menin in eukaryotic cells, the acquisition of pcDNA3.1-mycHis(-)A / bMEN, specifically comprises the following steps:

[0032] 1. cDNA cloning of bovine wild-type full-length MEN1 gene

[0033] The mammary gland tissues of Chinese Holstein cows in mid-lactation were collected, frozen and stored in liquid nitrogen immediately, and then total RNA was extracted with Trizol (Invetrogen, US.), and cDNA was synthesized with Superstcript III kit (Invitrogen, US.);

[0034] Primers were designed according to the complete mRNA sequence NM_001076161.2 of the bovine MEN1 gene published in Genebank, wherein, the sequence of the upstream primer (5'-atggggctgaaggctgcccagaaaacg-3'); in addition, protection bases g, Recognition site for restriction restriction of EcoRI and kozak sequence gccacc to enhance the expression efficiency of eukaryotic cells, so that the sequence of the upstream primer is 5'-g gaattc gccaccatggggctg...

Embodiment 2

[0049] Example 2 Expression detection of pcDNA3.1-mycHis(-)A / bMEN recombinant plasmid in bovine mammary gland epithelial cell MAC-T cell, Chinese hamster ovary cell CHO and mouse myoblast C2C12

[0050] Cell culture conditions and transfection: Bovine mammary gland epithelial cells MAC-T, Chinese hamster ovary cells CHO and mouse myoblast C2C12 cells were added with double antibody (1%) and 10% FBS (Gibico, US.) in DMEM ( Gibico TM , US.) medium, 37°C, 5% CO 2 After two weeks of culture under certain conditions, 24 hours before transfection, the culture medium without double antibody was coated on a 6-well plate at a density of 100 cells / well, and 2ml of DMEM medium containing 10% FBS was added to each well. After 24 hours, the cells were 50-80% confluent and transfected with liposomes (lipofectamine 2000 TM , Invitrogen, US.) The pcDNA3.1-mycHis(-)A / bMEN recombinant plasmid and the pcDNA3.1-mycHis(-)A empty plasmid were transfected into three kinds of cells respectively.

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Abstract

The invention relates to the animal genetics and the animal molecular cell biology, and provides an expression vector capable of efficiently expressing cow menin in eukaryotic cells. The expression vector capable of efficiently expressing cow menin in the eukaryotic cells provides necessary tools and means for the further research on the functions of the gene in the cells and the functional mechanism of the gene. The gene has expression in multiple organs in an organism and plays an important regulating role in regulating organism metabolism. According to the research on the functions and metabolic regulating and controlling pathways of the gene in a particular tissue or cell type, the fact that the gene is made to be excessively expressed under a specified condition by constructing the eukaryotic expression vector is an essential technological mean. The constructed efficient eukaryotic cell expression vector of the cow MEN1 gene (hereinafter referred to as bMEN1) can be used for providing necessary earlier research for the organism metabolic diseases of each tissue and organ of a cow, and even provides important tools and research means of a mode type research for the treatment of human diseases.

Description

technical field [0001] The invention relates to animal genetics and animal molecular cell biology, and provides an expression vector capable of efficiently expressing bovine menin in eukaryotic cells. Background technique [0002] The bovine MEN1 gene is highly homologous to human or mouse MEN1, and the homology of its encoded protein to human or mouse menin is as high as 98.69% and 96.73%, respectively. The results of human or mouse research imply that bovine MEN1 / menin plays an important role in regulating the normal metabolism of bovine (dairy cows), and it is speculated that its abnormal expression is related to bovine metabolic disorders (ketosis, acidosis, fatty liver, etc.) seizures are closely related. Human menin protein is the expression product of multiple endocrine neoplasia type 1 (MEN1) gene, which encodes 610 amino acids and is a nuclear protein expressed in multiple tissues. The key pathogenic gene MEN1 gene is located at the 11q13 locus of human chromosome...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12N15/66
Inventor 师科荣刘学王中华李洪辉杜红霞岳书俭殷彬林雪彦侯秋玲
Owner SHANDONG AGRICULTURAL UNIVERSITY
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