Sea anemone neurotoxin gene Sr6 and application thereof

A technology of neurotoxin and sea anemone, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of less neurotoxin in sea anemone, and achieve the effect of prolonging the effective refractory period and enhancing atrial contraction

Inactive Publication Date: 2005-03-16
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few domestic rese

Method used

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  • Sea anemone neurotoxin gene Sr6 and application thereof
  • Sea anemone neurotoxin gene Sr6 and application thereof
  • Sea anemone neurotoxin gene Sr6 and application thereof

Examples

Experimental program
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Embodiment 1

[0028] Example 1: Extraction and RT-PCR amplification of total RNA from tentacles of rose red and green sea anemone

[0029]Extraction of total RNA and synthesis of cDNA: the tentacles of rose red and green sea anemone were isolated, the total RNA of tentacles was extracted by guanidine isothiocyanate method, and the protein was removed by phenol / chloroform extraction. Obtain 100 μg total RNA of venomous glands, its A 260 / A 280 =2.03, two clear bands of 18s and 28s can be seen by 1% formaldehyde denaturing gel electrophoresis, and the ratio is >1 (see Figure 2), indicating that the integrity of the total RNA is good.

[0030] 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 cDNA first strand product.

[0031] Primer design and RT-PCR amplification: According to the amino acid s...

Embodiment 2

[0036] Example 2: Determination and analysis of recombinant sea anemone neurotoxin gene sequence

[0037] The recovered electrophoresis products were connected to the T vector, transformed into DH5 α Escherichia coli, and the recombinant clones were selected for sequencing. A total of 25 clones were detected, and Blast homology analysis showed that these 25 clones are all neurotoxin gene sequences, the gene length is 141bp, each encodes a toxin protein of 47 amino acids, and one of the toxin proteins is numbered Sr-p6 , which is not completely identical to the amino acid sequence of the reported sea anemone neurotoxin protein, and is a new toxin protein.

[0038] At present, there are more than 20 kinds of sea anemone neurotoxins reported, the number of amino acids is between 46-49 (in addition, there are several toxins with amino acid residues between 27-31, called short-chain neurotoxins), of which 12 amino acids are conserved . According to their amino acid sequences, the...

Embodiment 3

[0041] Example 3: Construction of recombinant sea anemone neurotoxin expression plasmid

[0042] A pair of primers were synthesized according to the sequences at both ends of the Sr6 gene, the upstream primers contained Kpn I and Prescission Protease cleavage sites, and the downstream primers contained BamH I cleavage sites.

[0043] Upstream primer (B1): 5'CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCC GGG GTG

[0044] Kpn I Precision Protease site

[0045] CCATGTTTG TGC GAC 3';

[0046] Downstream primer (B2): 5'CGC GGA TCC TTA TTA CTT CTT GCA GCA CCA GCC AAT A3';

[0047] Bam H I

[0048] The pGEM-T Easy plasmid (purchased from Promega) containing the Sr6 gene was used as a template, and B1 and B2 were used as primers for PCR amplification to obtain a specifically amplified single band with a product size of about 200 bp. The PCR amplification product was first cloned into the BSK plasmid with the combination ...

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Abstract

The invention relates to actinocongestin gene Sr6 and its coded protein, and also the uses of the said protein in preparation of drugs for cardiovascular disease. The invention employs RT-PCR method, extracts sequence 1 from total RNA of tentacles of sagartia rosea ( sequence 1 indicates) the protein coded by the gene( sequence 2 indicates) Recombinant actinocongestin protein in the invention has cardiac and anti-arrhythmia action, can prepare the cardiovascular disease drugs.

Description

technical field [0001] The invention relates to a sea anemone neurotoxin gene Sr6 and its coded protein, as well as the application of the protein in the preparation of drugs for treating cardiovascular diseases. Background technique [0002] Sea anemone (anthopleura), belonging to coelenterate (soelenterata), coral class (anthozoa), is a relatively primitive animal in the ocean. In the long-term biological evolution process, in order to resist predators and capture food, the nematocysts of sea anemone tentacles can secrete a variety of sea anemone toxins, basically polypeptide toxins, which have various physiological activities on humans and animals. Sea anemone toxins are divided into three categories according to their physiological functions: sea anemone neurotoxins, sea anemone cytolysins, and sea anemone potassium channel inhibitors. [0003] Foreign studies on sea anemone neurotoxins began in the 1970s. In 1976, Shibata and others from the University of Hawaii in th...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P9/00C07K14/44C07K19/00C12N15/10C12N15/30C12N15/70C12P19/34
Inventor 徐安龙陈慧萍王磊董美玲杨文利姜孝玉涂洪斌
Owner SUN YAT SEN UNIV
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