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Goat mTOR gene cDNA encoding zone 3í» terminal nucleotide sequence

A technology of nucleotide sequence and coding region, applied in the field of cDNA, can solve the problems of no goat mTOR gene cDNA nucleotide sequence, no mTOR research report, etc.

Inactive Publication Date: 2008-08-20
旭日干 +2
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  • Abstract
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  • Application Information

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Problems solved by technology

[0008] So far, the research on the regulatory role of mTOR in the growth and proliferation of mammalian cells has been carried out in human, mouse, bovine and other mammalian cells and many achievements have been made, but there is no research report on mTOR in goat cells. There is no report on the cDNA nucleotide sequence of goat mTOR gene and its corresponding amino acid sequence

Method used

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  • Goat mTOR gene cDNA encoding zone 3í» terminal nucleotide sequence
  • Goat mTOR gene cDNA encoding zone 3í» terminal nucleotide sequence
  • Goat mTOR gene cDNA encoding zone 3í» terminal nucleotide sequence

Examples

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Embodiment 1

[0029] Embodiment 1: Cloning of the cDNA coding region 3' end fragment of goat mTOR gene

[0030] In order to clone the cDNA containing the 3' end coding region of mTOR from goat testicular tissue cells, according to the nucleotide sequences (NM004958, NM019906, NM020009) disclosed in Figure 1 (A, B, C), first prepare the cDNA that allows amplification of 612bp PCR degenerate primers, that is, the upstream primer 5'-GAC CGT / G CTG AGT GGG / A AAG-3' and the downstream primer 5'-TTA CCA GAA A / GGGG / ACA CCA G-3'. Then add a BamH I restriction site at the 5' end of the upstream primer, and add an Xba I restriction site at the 5' end of the downstream primer, that is, the upstream primer P15'GC GGATCC GAC CGT / G CTG AGT GGG / AAAG 3' , downstream primer P25'GC TCTAGATTA CCA GAAA / GGG G / ACA CCA G 3'.

[0031] In order to clone the cDNA of mTOR by RT-PCR, goat total RNA was isolated from goat testis tissue, and the total RNA of goat testis tissue was extracted by using TaKaRa Total RNA Ext...

Embodiment 2

[0036] Embodiment 2: the nucleotide sequence of the cDNA of goat mTOR gene

[0037] Fig. 2 shows the nucleotide sequence (SEQ ID NO: 1) of the cDNA clone obtained in Example 1, and the amino acid sequence (SEQ ID NO: 2) deduced therefrom. In Fig. 2, the sequence shown is the nucleotide sequence of mTOR, which includes a 612 bp nucleotide sequence at the 3' end of the cDNA coding region, encoding 203 amino acids forming a mature protein with an inferred molecular weight of 23.1 KDa.

[0038] Figure 6 (A, B, C) shows the comparison of the nucleotide sequence of goat mTOR gene cDNA with the nucleotide sequence of human (NM004958), rat (NM019906) and mouse (NM020009) mTOR cDNA. Show that: 1) the nucleotide sequence of goat mTOR gene cDNA has 93% homology with the human mTOR gene cDNA nucleotide sequence; it has 90% homology with the rat mTOR gene cDNA nucleotide sequence; It has 89% homology with mouse mTOR nucleotide sequence. The amino acid sequence deduced from this nucleotid...

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Abstract

The invention relates to a cDNA which encodes mTOR protein and is separated from a goat testicle cell and the corresponding amino acid sequence. The invention finds a conservation region by comparing the nucleotide sequences of the known human, rats and mice mTOR genes and then designs a pair of degenerate primers for amplifying the 3' end fragment of a cDNA coding area of a goat mTOR gene by RT-PCR, the primers are used for amplifying the specific fragment by the PT-PCR method, the nucleotide sequence of 612bp of the 3' end of the cDNA coding area of the goat mTOR gene and the corresponding amino acid sequence are obtained after sequencing. The obtained 612bp nucleotide sequence can be further used for full-length cDNA cloning of the goat mTOR gene and detecting the tissue expression specificity of the mTOR gene by the RT-PCR or the hybridization method, which can also be used for preparing an antibody for detecting the mTOR after recombinant expression.

Description

technical field [0001] The present invention relates to cDNA encoding mTOR protein isolated from goat testicular cells, more specifically relates to the 3' terminal nucleotide sequence of the cDNA coding region encoding mTOR protein and its corresponding amino acid sequence. Background technique [0002] TOR (target of rapamycin) belongs to the PIKK superfamily, functions as a Ser / Thr kinase, and was first discovered in yeast. In mammalian cells, it is called mTOR (the mammalian target of rapamycin) or FRAP, RAFT, RAPT, and the gene was first isolated and cloned in 1994. Unlike yeast, there is only one TOR gene in mammalian cells, but the mTOR protein forms two complexes with different components, called mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin (rapamycin), mediates the signal transduction process related to nutritional factors, regulates the growth and proliferation of cells, and its function and regulation mechanism are well understood; mTORC2 is sensitive to r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/47
Inventor 旭日干吴应积王志钢
Owner 旭日干
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