Amphioxus dimethyl arginine hydrolase AmphiDDAH gene and its application

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 invention relates to Amphioxus dimethylarginine hydrolase (AmphiDDAH) gene and protein P encoded by it 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 Chordata and cephalochordate, is the closest living invertebrate to vertebrates, and is also the model of vertebrate ancestors. It is the most primitive species in the ocean. of animals. During the development of amphioxus embryos, in order to improve their own immunity and the differentiation and formation of tissues and organs, amphioxus embryos can produce DDAH, hydrolyze asymmetric dimethylarginine (ADMA), and enhance the synthesis of nitric oxide Enzyme (NOS) activity, thereby regulating the content of nitric oxide (NO) in the body.

[0003] In 1989, Ogawa first isolated DDAH in mouse liver by biochemical extraction method. T...

Examples

Embodiment 1

[0025] Example 1: Extraction of Total RNA and Synthesis of cDNA in Amphioxus neurula

[0026] Extraction of total RNA and cDNA synthesis: Collect amphioxus neurula, use Trizol reagent to extract total RNA of neurula, extract protein with phenol / chloroform, and obtain total RNA of amphioxus neurula, the A 260 / A 280 =1.828, two clear bands of 18s and 28s can be seen by 1% formaldehyde denaturing gel electrophoresis, the ratio>1 (see Figure 2), indicating that the total RNA has better integrity and higher purity. Take 1ug of total RNA, with SMART III olignuclotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII / 3'PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3′) was reverse-transcribed to synthesize the first strand to obtain 10 μl of one-strand eDNA product.

[0027] The 2ulcDNA one-strand product was taken, and the second-strand cDNA was synthesized by LD-PCR with 5'PCR Primer (5'-TTCCACCCAAAGCAGTGGTATCAACGCAGAGTGG-3') and 3'PCR Primer (5'-GTATCGATGCC...

Embodiment 2

[0028] Example 2: Determination and analysis of amphioxus neurula AmphiDDAH gene sequence

[0029] The second-strand cDNA was connected to the pcDNA3.0 vector, transformed into DH5α Escherichia coli, and the recombinant clone was selected for sequencing. Blast homology analysis showed that the EST sequence encoding the full-length AmphiDDAH gene was obtained, and the length of the gene was 822bp, encoding a P of 274 amino acids. DDAH protein, is a new DDAH protein.

[0030] Using the ClustalW analysis software, the reported DDAH protein sequences of different species were compared, and the results are shown in Figure 1.

[0031] It can be seen from Figure 1 that the three active sites of DDAH are relatively conserved in different species.

Embodiment 3

[0032] Example 3: Recombinant P DDAH Construction of expression plasmids

[0033] A pair of primers were synthesized according to the sequences at both ends of the AmphiDDAH gene, the upstream primers contained Kpn I and PrescissionProtease cutting sites, and the downstream primers contained Not I cutting sites.

[0034] Upstream primer (P1):

[0035] 5'CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCC ATG GCG

[0036] KpnI Precision Protease site

[0037] GAT TTC TGT TGG AAT TT3';

[0038] Downstream primer (P2):

[0039] 5'ATAAGAAT GCGGCCGC TTA CTA GAA CAG CAG AGA TTG GCA CGT C 3';

[0040] Not I

[0041] 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 specifically amplified single band was obtained with a product size of about 800 bp. The PCR amplified product was cloned into the prokaryotic fusion expression vector pETTRX to obtain th...

technical field

[0001] The invention relates to Amphioxus dimethylarginine hydrolase (AmphiDDAH) gene and protein P encoded by it 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 Chordata and cephalochordate, is the closest living invertebrate to vertebrates, and is also the model of vertebrate ancestors. It is the most primitive species in the ocean. of animals. During the development of amphioxus embryos, in order to improve their own immunity and the differentiation and formation of tissues and organs, amphioxus embryos can produce DDAH, hydrolyze asymmetric dimethylarginine (ADMA), and enhance the synthesis of nitric oxide Enzyme (NOS) activity, thereby regulating the content of nitric oxide (NO) in the body.

[0003] In 1989, Ogawa first isolated DDAH in mouse liver by biochemical extraction method. T...

Examples

Embodiment 1

[0025] Example 1: Extraction of Total RNA and Synthesis of cDNA in Amphioxus neurula

[0026] Extraction of total RNA and cDNA synthesis: Collect amphioxus neurula, use Trizol reagent to extract total RNA of neurula, extract protein with phenol / chloroform, and obtain total RNA of amphioxus neurula, the A 260 / A 280 =1.828, two clear bands of 18s and 28s can be seen by 1% formaldehyde denaturing gel electrophoresis, the ratio>1 (see Figure 2), indicating that the total RNA has better integrity and higher purity. Take 1ug of total RNA, with SMART III olignuclotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII / 3'PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3′) was reverse-transcribed to synthesize the first strand to obtain 10 μl of one-strand eDNA product.

[0027] The 2ulcDNA one-strand product was taken, and the second-strand cDNA was synthesized by LD-PCR with 5'PCR Primer (5'-TTCCACCCAAAGCAGTGGTATCAACGCAGAGTGG-3') and 3'PCR Primer (5'-GTATCGATGCC...

Embodiment 2

[0028] Example 2: Determination and analysis of amphioxus neurula AmphiDDAH gene sequence

[0029] The second-strand cDNA was connected to the pcDNA3.0 vector, transformed into DH5α Escherichia coli, and the recombinant clone was selected for sequencing. Blast homology analysis showed that the EST sequence encoding the full-length AmphiDDAH gene was obtained, and the length of the gene was 822bp, encoding a P of 274 amino acids. DDAH protein, is a new DDAH protein.

[0030] Using the ClustalW analysis software, the reported DDAH protein sequences of different species were compared, and the results are shown in Figure 1.

[0031] It can be seen from Figure 1 that the three active sites of DDAH are relatively conserved in different species.

Embodiment 3

[0032] Example 3: Recombinant P DDAH Construction of expression plasmids

[0033] A pair of primers were synthesized according to the sequences at both ends of the AmphiDDAH gene, the upstream primers contained Kpn I and PrescissionProtease cutting sites, and the downstream primers contained Not I cutting sites.

[0034] Upstream primer (P1):

[0035] 5'CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCC ATG GCG

[0036] KpnI Precision Protease site

[0037] GAT TTC TGT TGG AAT TT3';

[0038] Downstream primer (P2):

[0039] 5'ATAAGAAT GCGGCCGC TTA CTA GAA CAG CAG AGA TTG GCA CGT C 3';

[0040] Not I

[0041] 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 specifically amplified single band was obtained with a product size of about 800 bp. The PCR amplified product was cloned into the prokaryotic fusion expression vector pETTRX to obtain th...

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