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Polypeptide human rudder chain dehydrogenase 9.46 and polynucleotide for encoding polypeptide

A short-chain dehydrogenase, polynucleotide technology, applied in oxidoreductase, anti-enzyme immunoglobulin, botanical equipment and methods, etc., can solve neuromuscular dysfunction, growth retardation and other problems

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

Deficiency in this enzyme results in neuromuscular dysfunction and growth retardation

Method used

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  • Polypeptide human rudder chain dehydrogenase 9.46 and polynucleotide for encoding polypeptide
  • Polypeptide human rudder chain dehydrogenase 9.46 and polynucleotide for encoding polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Cloning of human short-chain dehydrogenase 9.46

[0082] Total RNA was extracted from human fetal brain by one-step method of guanidine isothiocyanate / phenol / chloroform. 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. Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of the pBSK(+) vector (product of Clontech Company), transform DH5α, and the bacteria formed a cDNA library. The sequences of the 5' and 3' ends of all clones were determined using Dye terminate cycle reaction sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 5017C07, was a new DNA. The insert cDNA fragment contained in this clone was det...

Embodiment 2

[0083] Embodiment 2: Cloning the gene encoding human short-chain dehydrogenase 9.46 by RT-PCR method

[0084] The total RNA of fetal brain cells was used as a template, and oligo-dT was used as a primer to carry out reverse transcription reaction to synthesize cDNA. After purification with a Qiagene kit, PCR amplification was performed with the following primers:

[0085] Primer1: 5'-GCTCCTTCTGAATGCTCTGGGGCA-3'

[0086] Primer2: 5'-GCACGGCTGCGAGAAGACGAAGCT-3'

[0087] Primer1 is the forward sequence starting from the 1bp at the 5' end of 1;

[0088] Primer2 is the 3' reverse sequence in 1.

[0089] The conditions of the amplification reaction: 50mmol / L KCl, 10mmol / L Tris-Cl, (pH8.5), 1.5mmol / L MgCl in a reaction volume of 50μl 2 , 200 μmol / L dNTP, 10 pmol primer, 1 U of Taq DNA polymerase (product of Clontech Company). On a PE9600 DNA thermal cycler (Perkin-Elmer), the reaction was performed for 25 cycles under the following conditions: 94°C for 30 sec; 55°C for 30 sec; 72...

Embodiment 3

[0090] Example 3: Northern blot analysis of human short-chain dehydrogenase 9.46 gene expression:

[0091] 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, use 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (DH4.0) to homogenate the tissue, add 1 times the volume of phenol and 1 / 5 volume of chloroform-isoamyl alcohol (49:1 ), mixed and centrifuged. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 20 µg of RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transfer to nitrocellulose membrane. with α- 32 P dATP prepared by random primer method 32 P-labeled DNA probes. T...

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Abstract

A novel polypeptide-human short-chain dehydrogenase 9.46, 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, cardiovascular disease, immunopathy, etc. the antagon of said polypeptide and its medical action, and the application of said polynucleotide are disclosed.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide-human short-chain dehydrogenase 9.46, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique [0002] Short-chain dehydrogenases have a large protein family with many members such as pyruvate dehydrogenase, 15-hydroxyprostaglandin dehydrogenase (NAD + ), alcohol dehydrogenase, carbonyl dehydrogenase, retinol dehydrogenase (NADP + ), 3α / 20β-hydroxysteroid dehydrogenase, 11β-hydroxysteroid dehydrogenase, etc., and are widely distributed in animals, plants, yeast, bacteria and other organisms, participating in the metabolic cycle. [0003] Most short-chain dehydrogenase members are dimers and tetramers, with four highly conserved characteristic motifs: the catalytic active site of the enzyme is near the C-termi...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N9/02C12N15/53C12N15/63
Inventor 毛裕民谢毅
Owner BIOWINDOW GENE DEV INC
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