Natriuretic peptide, as well as gene and use thereof

A technology of natriuretic peptides and genes, applied in the field of natriuretic peptides, can solve the problems of few research reports on natriuretic peptides

Active Publication Date: 2014-05-28
叶亮 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with ANP, BNP and CNP, there are fewer research reports on other natriuretic peptides

Method used

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  • Natriuretic peptide, as well as gene and use thereof
  • Natriuretic peptide, as well as gene and use thereof
  • Natriuretic peptide, as well as gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: cDNA cloning and gene analysis of GNP

[0039] Using Invitrogen's Rapid mRNA Extraction Kit to extract mRNA from the freshly isolated snake venom tissue of the East African Green Mamba, and then use Clontech's SMART TM The cDNA library construction kit is used to construct the cDNA library of snake venom gland tissue. The kit uses 5' and 3' RACE technology for rapid cloning of cDNA ends.

[0040] Primer for 5'RACE: SEQ ID NO: 3

[0041] 5'-TGGTCGATCTTGTGGCCGAAGCAGCCAT-3'.

[0042] Primer for 3'RACE: SEQ ID NO: 4

[0043] 5'-TAGACTCGCGTCGTATGAAGGGGCTGGA-3'.

[0044] The RACE product was recovered by cutting the gel to obtain a full-length cDNA sequence of a novel natriuretic peptide, namely GNP of the present invention.

[0045] The full-length cDNA sequence and deduced amino acid sequence of GNP can be found in figure 1 and sequence listing. The full length of the cDNA is 718bp, and its nucleotide sequence is shown in SEQ ID NO: 2. Among them,...

Embodiment 2

[0062] Example 2: Artificial synthesis and analysis of GNP protein

[0063] The inventor artificially synthesized the GNP protein according to the analysis results in Example 1, and entrusted Shanghai Institute of Biochemistry to conduct molecular weight analysis (mass spectrometry), purity analysis (HPLC method), and N-terminal sequencing test analysis on the synthetic polypeptide samples provided by the inventor. , The presence or absence of disulfide bonds in the polypeptide molecule and the analysis of the location of disulfide bonds.

[0064] 1. Molecular weight analysis

[0065] Sample state: solid.

[0066] Detection method: mass spectrometry.

[0067] The main instrument used: mass spectrometer.

[0068] Analyzing test results:

[0069] Mass spectrum molecular weight 3690.5146 3690.5146

[0070] Theoretical molecular weight 3689.7900 3689.79.

[0071] 2. Purity analysis

[0072] Sample state: solid.

[0073] Detection basis: Chinese Pharmacopoeia 2010 edition...

Embodiment 3

[0111] Example three: cGMP secretion experiment on PC12 cells

[0112] Experimental materials: PC12 cells (a kind of rat adrenal gland cells), purchased commercially.

[0113] According to previous reports, in PC12 cells, the ratio of NPRA (natriuretic peptide receptor A) to NPRB (natriuretic peptide receptor B) is 3:1, that is, NPRA is dominant.

[0114] In this experiment, the stimulatory effects of five natriuretic peptide family members including GNP on cGMP secretion in PC12 cells were studied. Among them, GNP is from the artificially synthesized sample in Example 2, and the other four natriuretic peptides are all from commercial purchases or gifts from scholars.

[0115] Experiment 1: Culture PC12 cells on a 24-well culture plate to 1×10 per well 6 cells, add 1×10 -6 ANP and GNP of M. At each time point, culture fluid from 6 different wells was taken out for cGMP concentration analysis (by immunoassay).

[0116] Figure 2A and Figure 2B ANP (1 × 10 -6 M) with ...

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Abstract

The invention relates to a natriuretic peptide from a snake and a gene encoding the polypeptide. The natriuretic peptide disclosed by the invention is the natriuretic peptide from dendroaspis angusticeps. The natriuretic peptide disclosed by the invention is the brand new natriuretic peptide and a new member in a natriuretic peptide family, and is further called as GNP, namely G type natriuretic peptide. The invention further provides a preparation method of the recombinant natriuretic peptide. The natriuretic peptide provided by the invention comprises the recombinant natriuretic peptide and an artificially synthesized natriuretic peptide, which can be applied to preparation of medicaments for treating patients with acute heart failure, acute decompensated heart failure, acute myocardial infarction after coronary intervention, chronic heart failure, and acute anterior wall myocardial infarction with systolic heart failure of old people.

Description

technical field [0001] The present invention relates to a natriuretic peptide, in particular to a natriuretic peptide from snakes, and a gene encoding the polypeptide. Background technique [0002] Natriuretic peptides (Natriuretic Peptide, NP) refer to a class of peptides with strong biological activities such as sodium excretion, diuresis, and vasodilator. These polypeptides have genetic homology and the same receptor system. [0003] Currently, natriuretic peptides obtained from mammals include: atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide. Atrial natriuretic peptide is mainly secreted by atrial myocytes, brain natriuretic peptide is mainly secreted by ventricular myocytes, C-type natriuretic peptide is mainly secreted by vascular endothelial cells and exerts vasodilator and antiproliferative effects locally. [0004] In 1981, Canadian scientist de Bold found that rat heart extract had strong natriuretic and diuretic effects. A y...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/575C12N15/16C12N15/63C12N1/21C12P21/02A61K38/22A61P9/04A61P9/10
Inventor 叶亮
Owner 叶亮
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