Cloning, Expression and bioactivity of new actinion toxin

An amino acid and technology in the sequence table, applied in the field of protein structure, can solve the problems of small detoxification, difficulty in collecting venom, and low production of sea anemones, etc., and achieve enhanced atrial contraction, high application prospects and industrial development value, strong The effect of promoting contraction

Inactive Publication Date: 2003-05-14
北京博奥环宇生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Because sea anemones produce less in my country's sea areas, and their detoxification is very small, it is difficult to collect enough venom. Practical application and other aspects bring certain difficulties

Method used

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  • Cloning, Expression and bioactivity of new actinion toxin
  • Cloning, Expression and bioactivity of new actinion toxin
  • Cloning, Expression and bioactivity of new actinion toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example One The extraction and RT-PCR amplification of the tentacle total RNA

[0046] Extraction of total RNA and synthesis of cDNA: Isolate the tentacles of a sea anemone lateralis, extract the total RNA of the tentacles by guanidine isothiocyanate method, and remove the protein by phenol / chloroform extraction. Obtain 100 μg of total RNA from the venomous gland, and its A 260 / A 280 = 2.03, two clear bands of 18s and 28s can be seen through 1% formaldehyde denaturing gel electrophoresis, the ratio> 1, and several specific RNA bands (see figure 1 ), indicating that the integrity of total RNA was good. 5 μg of RNA was reverse-transcribed with Oligo-dT to synthesize the first-strand cDNA, and 20 μl of the first-strand cDNA product was obtained.

[0047] Primer design and RT-PCR amplification were based on: According to the amino acid sequences at both ends of Ap-B, oligo primer design software was used to assist analysis, and two degenerate primers were designed. Amin...

Embodiment 2

[0049] Add 1 μl cDNA single strand to every 50 μl PCR reaction volume as a template, perform PCR amplification, and detect by electrophoresis. It is found that the expected specific amplification band appears around 150 bp (see figure 2 ), recycle the tape. Example 2 Determination and Analysis of Recombinant Sea Anemone Neurotoxin Gene Sequence

[0050] The recovered electrophoresis products were connected to the T vector, transformed into DH5α Escherichia coli, and the recombinant clones were selected for sequencing. A total of 17 clones were determined, and Blast homology analysis showed that 8 of them are neurotoxin gene sequences, and the length of these 8 toxin genes is 141bp, encoding a toxin protein with a length of 47 amino acids. The toxin protein number is Sea anemone toxin-su, whose amino acid sequence is not identical with the reported sea anemone neurotoxin protein, is a new toxin protein.

[0051] At present, there are more than 20 kinds of sea anemone neuroto...

Embodiment 3

[0059] In the PCR amplification reaction, there will be about 10-4 mismatches with ordinary Taq enzymes. Since the target fragment is small, the number of PCR cycles does not exceed 30, which reduces the probability of wrong inclusion. From the experimental results, This sequence appeared in multiple clones, indicating that the results of PCR have high reliability. Example 3 Construction of Recombinant Sea Anemone Neurotoxin Expression Plasmid

[0060] A pair of primers were synthesized according to the sequences at both ends of the su gene, the upstream primers contained Kpn I and Prescission Protease cleavage sites, and the downstream primers contained BamH I cleavage sites. Upstream primer (B1): 5'CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCC GGG GTT

[0061] Kpn I Precision Protease site

[0062] CCG TGT TTG TGT GAC 3' downstream primer (B2): 5'CGC GGA TCC TTA TTA CTT CTT GCA GCA CCA GCC AAT G3’

[0063] Bam H I

[...

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Abstract

By means of constituting Anthopleura sp tentacle cDNA library and DNA sequencing and designing degenerate primer based on the end conservative amino acid sequences of actinia neurotoxin protein, one new kind of actinia polypeptide neurotoxin gene, named su, is separated via RT-PCR process. The new gene has length of 141 bp and encodes 47 amino acid mature peptides. The expressed protein has ioelectric point of 7.9 and molecular weight of 4800 dalton. Cloning su in the trxA gene 3' end of thioredoxin gene fusion expression vector pETTRX, fusion gene is constituted and the fusion gene exists incolibacillus in intracellar soluble form with expression amount up to 100-120 mg/L. Through Ni-Chelating affinity chromatographic analysis, proteinase 3C cutting and gel filtering, recombinant actinia neurotoxin protein with purity over 95% is obtained with the yield of 10-50 mg/L.

Description

technical field [0001] The present invention relates to the cloning, expression and biological activity of the new gene of sea anemone poison, more specifically, relates to a kind of new gene isolated from the total RNA of the tentacle of the sea anemone, using the method of genetic engineering in the high-efficiency Escherichia coli expression system Express and purify the biologically active recombinant neurotoxin protein and the drug for treating cardiovascular system diseases composed of this protein. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/614A61P9/00C12N15/12C12N15/63C12N15/70
Inventor 徐安龙刘文华彭文烈卫剑文彭立胜陈琪
Owner 北京博奥环宇生物技术有限公司
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