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Polypeptide-human HADH dehydrogenase substructured protein I-9.25 and polynucleotide for encoding polypeptide

A polynucleotide, dehydrogenase sub-technology, applied in the field of polynucleotide sequence, polynucleotide and polypeptide preparation, can solve the problems of influence, abnormal expression of subunits, etc.

Inactive Publication Date: 2004-08-11
BIOWINDOW GENE DEV INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two sequence fragments may be the active center of subunit I of the enzyme complex, and their mutation will lead to abnormal expression of the subunit, which cannot be combined with coenzyme Q normally, thereby affecting its role in the respiratory chain

Method used

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  • Polypeptide-human HADH dehydrogenase substructured protein I-9.25 and polynucleotide for encoding polypeptide
  • Polypeptide-human HADH dehydrogenase substructured protein I-9.25 and polynucleotide for encoding polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Cloning of human NADH dehydrogenase substructure protein I-9.25

[0085] Total RNA from human fetal brain was extracted by one-step guanidine isothiocyanate / phenol / chloroform method. Poly(A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (product of Qiegene). 2ug poly(A)mRNA was reverse transcribed to form cDNA. Using Smart cDNA cloning kit (purchased from Clontech), the cDNA fragment was directionally inserted into the multiple cloning site of pBSK(+) vector (product of Clontech Company), transformed into DH5α, and the bacteria formed a cDNA library. The 5' and 3' ends of all clones were sequenced with Dye terminate cycle reaction sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). The determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones, 5029H12, was a new DNA. Bidirectional determination of the ins...

Embodiment 2

[0086] Example 2: Cloning of the gene encoding human NADH dehydrogenase substructural protein I-9.25 by RT-PCR

[0087] The total RNA of fetal brain cells was used as the template, and oligo-dT was used as the primer for reverse transcription reaction to synthesize cDNA. After purification with Qiagene kit, PCR amplification was performed with the following primers:

[0088] Primer1: 5'-GGACAGAGTTTACATTGATACTCA-3'

[0089] Primer2: 5'-CATAGGCCGAGGCGGGCCGACATG-3'

[0090] Primer1 is a forward sequence located at the 1 bp of the 5' end of 1;

[0091] Primer2 is the 3' reverse sequence of 1.

[0092] Amplification reaction conditions: 50 mmol / L KCl, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl in a 50 μl reaction volume 2 , 200μmol / L dNTP, 10pmol primer, 1U Taq DNA polymerase (product of Clontech company). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94°C for 30 sec; 55°C for 30 sec; 72°C for 2 min. In...

Embodiment 3

[0093] Example 3: Northern blot analysis of the expression of human NADH dehydrogenase substructural protein I-9.25 gene:

[0094] Total RNA was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159]. The method includes acid guanidine thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH 4.0), and 1 volume of phenol and 1 / 5 volume of chloroform-isoamyl alcohol (49:1) were added. ), mixed and centrifuged. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain the RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 20 μg RNA was used for electrophoresis on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2 M formaldehyde. Then transfer to nitrocellulose membrane. with α- 32 P dATP was prepared by random...

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Abstract

A novel polypeptide-human NADH dehydrogenase substructure protein I-9.25, the polynucleotide for coding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide in treating diseases, such as cancer, HIV infection, immunopathy, etc. the antagon of said polypeptide and its medical action, and the application of said polynucleotide are disclosed.

Description

technical field [0001] The present invention belongs to the field of biotechnology, in particular, the present invention describes a novel polypeptide, NADH dehydrogenase substructure protein I-9.25, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to preparation methods and applications of such polynucleotides and polypeptides. Background technique [0002] Respiratory chain NADH dehydrogenase (also known as complex I or NADH-coenzyme Q oxidoreductase) is a polymerase complex present in the inner mitochondrial membrane, chloroplasts and cyanobacteria. In cyanobacteria, it is also used as an NADH-plastoquinone oxidoreductase. The enzyme complex consists of 25-30 polypeptide chain subunits, 15 of which are present on the membrane, and 7 of these 15 subunits are represented by mitochondrial and chloroplast genomes in many different species coding. The conserved subunit encoded by these organelles is called subunit I (encoded by the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N9/02C12N15/53C12N15/63
Inventor 毛裕民谢毅
Owner BIOWINDOW GENE DEV INC
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