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Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fatty liver in lipid biosynthesis

A technology of isocitrate dehydrogenase and biosynthesis, which can be applied in gene therapy, microbiological measurement/testing, medical preparations containing active ingredients, etc., and can solve the problems of lack of IDPc metabolic function, etc.

Inactive Publication Date: 2004-08-25
TG LIFE IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, there are no reports on the metabolic function of IDPc

Method used

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  • Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fatty liver in lipid biosynthesis
  • Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fatty liver in lipid biosynthesis
  • Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fatty liver in lipid biosynthesis

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Experimental program
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preparation example Construction

[0047] 1. Preparation of fusion gene constructs

[0048] In order to permanently express the IDPc gene, the gene needs to be integrated into the genome of the animal. For this purpose, it is first necessary to construct recombinant constructs capable of expressing the gene of interest in mammals. In the resulting fusion gene construct, expression of the gene of interest is under the control of an appropriate promoter gene. The term "fusion gene construct" as used herein refers to a functional combination of genes used in a specific biotransformation, which mainly includes at least one structural gene, and at least one sequencer for controlling the expression of the structural gene. Functional regulatory elements.

[0049] Typically, cis-acting regulatory elements can be in the form of promoters, enhancers, introns, 5'-UTRs (untranslated regions), and 3'-UTRs. In the fusion gene construct, the cis-acting regulatory element can be located at any site within a distance of le...

Embodiment 1

[0076] Example 1: Isolation and sequencing of IDPc gene

[0077] 1-1. Isolation of mouse IDPc cDNA

[0078] A probe for identifying mouse IDPc cDNA was prepared using the recently reported rat IDPc cDNA (Jennings et al., J. Biol. Chem., 169, 21328-23134, 1994). The sense primer listed in the No. 1 sequence was synthesized based on nucleotides 532-550 of the rat IDPc cDNA gene, and the antisense primer listed in the No. 2 sequence was synthesized based on nucleotides 1263-1245. Separately, mRNA isolated from rat liver was converted into cDNA by using reverse transcriptase. Using these primers for PCR, first pre-denaturation at 94°C for 4 minutes; then perform 25 cycles: denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute, and extension at 72°C for 2 minutes; finally further at 72°C For 10 min extension, 100 ng of cDNA library was used as template. As a result, a DNA sequence of 0.8 kb was amplified. This PCR product was cloned into pCR II (Invitrogen Co.). ...

Embodiment 2

[0087] Embodiment 2: Construction of the cell line transformed with IDPc gene

[0088] 2-1: Construction of recombinant retroviral vector for expressing IDPc gene

[0089] Regarding the expression of the IDPc gene in cells, the IDPc cDNA obtained from Example 1 was subcloned in sense and antisense orientations into the retroviral vector pLNCX (Miller, A.D. and Rosman, G.T., Biotechniques, 7, 980-990, 1989).

[0090] Both ends of the IDPc cDNA were cut with ClaI, and the cut DNA was ligated into a retroviral vector. The recombinant plasmid was introduced into E. coli DH5α and amplified by culturing the microorganism. The orientation of the cloned insert was identified by digestion with restriction enzymes. In the resulting recombinant vector construct, such as figure 1 Sense or antisense expression of IDPc cDNAs was directed by using the cytomegalovirus promoter as shown. The recombinant vector with IDPc cDNA inserted in the sense direction determined by restriction en...

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Abstract

The present invention relates to a cytosolic isocitrate dehydrogenase, its gene, and its use in the treatment of obesity, hyperlipidemia, and fatty liver. The expression of the IDPc gene and the concomitant increase in IDPc level bring about an increase in the cellular level of NADPH, which causes the lipid deposition in adipocytes, leading to obesity and fatty liver. A decrease in the cellular level of NADPH, resulting from the suppression of the gene expression of IDPc, has the effect of inhibiting the lipid deposition in adipocytes. Further, by taking advantage of the suppressive or inhibitory effects of isocitrate dehydrogenase inhibitors, pharmaceutically effective materials for the prophylaxis and treatment of obesity, hyperlipidemia and fatty liver can be developed.

Description

field of invention [0001] The present invention relates to an isocitrate dehydrogenase that catalyzes the production of NADPH necessary for the biosynthesis of lipids including fatty acids, squalene and cholesterol, and also relates to isocitrate dehydrogenase Use in the treatment of metabolic diseases, including obesity, hyperlipidemia and fatty liver. Likewise, the invention relates to the isocitrate dehydrogenase gene, fusion gene constructs containing the gene, transfectant cells containing the gene in their genome, and cells capable of continuously expressing isocitrate dehydrogenase throughout their lifespan. of transgenic animals. Background technique [0002] Isocitrate dehydrogenase, which participates in the TCA (tricarboxylic acid) cycle, catalyzes the oxidative decarboxylation of citric acid to α-ketoglutarate and simultaneously produces NADH or NADPH. [0003] In higher animals, isocitrate dehydrogenase isomerases can be divided into three groups according to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02A01K67/027A61K38/00A61K38/43A61K38/44A61K45/00A61K48/00A61P3/04A61P3/06C12N1/15C12N1/19C12N1/21C12N5/10C12N9/04C12N9/99C12N15/09C12N15/53C12N15/63C12Q1/02C12Q1/32C12Q1/60G01N33/15G01N33/50
CPCC07K2319/00A01K2217/05C12N9/0006A61K38/00C12Y101/01041A61P3/04A61P3/06
Inventor 许太辚高豪振崔明淑郑运珠
Owner TG LIFE IND
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