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Amphioxus dimethyl arginine hydrolase AmphiDDAH gene and its application

A kind of amphioxus, gene technology, applied in the direction of hydrolase, application, genetic engineering, etc., can solve the problem of few research reports on DDAH and so on

Inactive Publication Date: 2008-07-23
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few research reports on DDAH in China.

Method used

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  • Amphioxus dimethyl arginine hydrolase AmphiDDAH gene and its application
  • Amphioxus dimethyl arginine hydrolase AmphiDDAH gene and its application
  • Amphioxus dimethyl arginine hydrolase AmphiDDAH gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Extraction of total RNA and cDNA synthesis of Amphioxus neuroblastus

[0025] Extraction of total RNA and cDNA synthesis: Collect amphioxus neuroblasts, use Trizol reagent method to extract neuroblast total RNA, and phenol / chloroform extraction to remove protein to obtain amphioxus neuroblast total RNA, its A 260 / A 280 =1.828, two clear 18s and 28s bands can be seen by 1% formaldehyde denaturing gel electrophoresis, the ratio is more than 1 (see figure 2 ), indicating that the total RNA has good integrity and purity. Take 1ug of total RNA, use SMART III olignuclotide (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3′) and CDSIII / 3′ PCR primers (5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3') Reverse transcription is performed to synthesize the first strand, and 10 μl one-strand cDNA product is obtained.

[0026] Take 2ul cDNA one-strand product, and use 5'-PCR Primer (5'-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3') and 3'-PCR Primer (5'-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGG...

Embodiment 2

[0027] Example 2: Determination and analysis of AmphiDDAH gene sequence of amphioxus neuroblastus

[0028] The two-stranded cDNA was ligated to pcDNA3.0 vector, transformed into DH5α Escherichia coli, and selected recombinant clones for sequencing. Blast homology analysis showed that the EST sequence encoding the full-length AmphiDDAH gene was obtained. The length of the gene is 822 bp, which encodes 274 amino acids. DDAH Protein, is a new DDAH protein.

[0029]Using ClustalW analysis software to compare the reported DDAH protein sequences of different species, the results are as follows figure 1 Shown.

[0030] From figure 1 It can be seen that the three active sites of DDAH are relatively conserved in different species.

Embodiment 3

[0031] Example 3: Recombinant P DDAH Construction of expression plasmid

[0032] A pair of primers were synthesized based on the sequence of both ends of the AmphiDDAH gene. The upstream primer contained Kpn I and Prescission protease cleavage sites, and the downstream primer contained Not I cleavage sites.

[0033] Upstream primer (P1):

[0034] 5’CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCC ATG GCG

[0035] Kpn I Prescission protease cleavage site

[0036] GAT TTC TGT TGG AAT TT3’;

[0037] Downstream primer (P2):

[0038] 5’ATAAGAAT GCGGCCGC TTA CTA GAA CAG CAG AGA TTG GCA CGT C3’;

[0039] Not I

[0040] Using the pcDNA3.0 plasmid (purchased from Invitrogen) containing the AmphiDDAH gene as a template, and P1 and P2 as primers for PCR amplification, a specific amplified single band was obtained, and the size of the product was about 800 bp. The PCR amplification product was cloned into the prokaryotic fusion expression vector pETTRX to obtain the ...

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Abstract

The present invention relates to Amphioxus gene AmphiDDAH, its coded protein, and the application of the protein in preparing medicine for treating cardiac and cerebral vascular diseases and arthritis. By means of cDNA library constitution and large scale sequencing process, the present invention separates Amphioxus neurula to obtain the Amphioxus gene AmphiDDAH with the DNA sequence shown in <400>1 of the sequence list. The gene coded protein, PDDAH, has amino acid sequence shown in <400>2 of the sequence list. The recombinant Amphioxus protein PDDAH has NO creation regulating function and may be used in preparing medicine for treating cardiac and cerebral vascular diseases and arthritis.

Description

Technical field [0001] The present invention relates to Amphi DDAH (AmphiDDAH) gene and its encoded protein P DDAH , And the application of the protein in the preparation of medicines for treating cardiovascular diseases and arthritis. Background technique [0002] Amphioxus (Branchiostoma belcheri tsingtauense), belonging to the Chordata and Cephalochordate, is the invertebrate closest to vertebrates, and a model of the ancestors of vertebrates. It is a primitive animal in the ocean. Of animals. During the development of Amphioxus embryos, in order to improve their own immunity and the differentiation and formation of their tissues and organs, Amphioxus embryos can produce DDAH, hydrolyze asymmetric dimethylarginine (ADMA), and enhance nitric oxide synthesis Enzyme (NOS) activity, thereby regulating the content of nitric oxide (NO) in the body. [0003] In 1989, Ogawa first used biochemical extraction method to isolate DDAH from mouse liver. This enzyme is a single peptide chain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/14C07K14/435C12N15/70C12P21/02C07K1/14A61K38/17A61P19/02A61P9/00C12N15/12
Inventor 徐安龙徐斌曹宝华崔彩媚谢珍慧张文献董美玲
Owner SUN YAT SEN UNIV
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