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Preparation and application of South China sea conus textile conotoxin TxO10

A technology of conotoxin and brocade conus, applied in the directions of application, medical preparations containing active ingredients, botanical equipment and methods, etc.

Inactive Publication Date: 2012-09-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, conotoxins whose functions have been clarified at home and abroad only account for a small part of the entire toxin pool, but their physiological functions have been clearly understood

Method used

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  • Preparation and application of South China sea conus textile conotoxin TxO10
  • Preparation and application of South China sea conus textile conotoxin TxO10
  • Preparation and application of South China sea conus textile conotoxin TxO10

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Extraction of total RNA and toxin cDNA cloning of brocade conus venom vascular tissue

[0037] The extraction of total RNA refers to the method of Gibcol BRL company LS reagent instructions were carried out. LATaq DNA polymerase, 10×PCR Buffer, DNA ladder were purchased from TaKaRa Company, dNTP was purchased from Dingguo Company, pGEM-T Easy Vector Systems were purchased from Promega Company; PCR primers were synthesized by Invitrogen Company; other organic reagents were domestic analytical grade , purchased from Guangzhou Chemical Reagent Factory.

[0038] Take live snails, break the snail shell with a masonry hammer to expose the snail meat, carefully and quickly separate the poison tube tissue on ice, weigh it, quickly put it into liquid nitrogen and grind it fully, add 15 times the weight volume of LS reagent (that is, add 15ml to 1g of tissue), fully homogenize in an ice bath, and store in a -80°C refrigerator for extraction of total RNA. Take 50-1...

Embodiment 2

[0069] Example 2: South China Sea brocade toxin polypeptide TxO 10 Construction of fusion expression vector

[0070] 1) Plasmids and strains:

[0071] Escherichia coli (Escherichia coli) DH5α was purchased from Invitrogen and kept in our laboratory, its genotype is: DH5α: supE44Mac U169 hsdR17recA1endAl gyrA96thi-1relA1; Escherichia coli expression host strain BL21 (DE3) was purchased from Stratagene Company, the genotype is: BL21 (DE3): F-ompT hsdSB (r-B, m-B) dcm galλ (DE3).

[0072] 2) Reagents and other materials:

[0073] Restriction enzymes KpnI, NotI and T4 DNA ligase were purchased from TaKaRa Company; Taq DNA polymerase, 10×PCR Buffer and dNTP were purchased from Dingguo Company; Gel Extraction Kit and Plasmid Miniprep Kit were products of OMEGA BIOTEK Company; BCA TM Protein Assay Kit was purchased from PIERCE Company; Trans2K DNAMarker was purchased from TransGen Company; low molecular weight standard protein 14,400-108,000kD was purchased from Keygen Biotechno...

Embodiment 3

[0102] Example 3, Efficient prokaryotic expression of the brocade conotoxin polypeptide gene from the South China Sea of ​​China

[0103] 1) Expression of fusion protein

[0104] The sequenced correct recombinant vector pTRX-TxO 10 Transformed into Escherichia coli BL21 (DE3) to construct engineering strains. Pick a single colony of the engineered strain and inoculate it in Amp + In LB liquid enriched medium, shake culture overnight at 37°C as seed bacteria. The seed bacteria were inoculated in fresh Amp at a volume ratio of 1:50 + In LB-rich medium, 37°C was vigorously shaken and amplified until OD600 was about 0.6, adding IPTG to a final concentration of 0.1mmol / L, and adding 20% ​​glucose to a final concentration of 0.2%, and induced expression at 18°C ​​for 12 hours. After induction, centrifuge at 4°C and 10,000 rpm for 10 min, collect the bacteria, and resuspend with pre-cooled Lysis Buffer (50 mmol / L Tris, 500 mmol / L NaCl, 10 mmol / L imidazole, pH 7.0) at a ratio of 1...

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Abstract

The invention relates to a preparation method and application of a South China Sea conus textile conotoxin gene TxO10 and Coded polypeptide TxO10 thereof. The method obtains thetoxin gene TxO10 by cloning from a venom duct of the South China Sea conus textile conotoxin through construction of a cDNA library. The invention provides a preparation method for polypeptide, comprising the following steps: recombination expression vector pTRX-TxO10 is obtained through connecting the conotoxin gene TxO10 and a carrier pTRX-TxO10, the expression vector pTRX-TxO10 is transformed into host bacterium for culturing expressing in the host bacterium, and object protein-Polypeptide TxO10 is obtained through separating and purifying the expression product recombination fusion protein. The polypeptide TxO10 provided in the invention has the function of improving action potential of sciatic nerve trunk, and can be used for making tool medicine for neurobiology research.

Description

technical field [0001] The present invention relates to a polypeptide and its coding gene sequence, its preparation method and application. belongs to the field of biology. Background technique [0002] Cone snail (cone snail) is a kind of carnivorous animal living in shallow ocean waters. Conus. It is currently estimated that there are about 500-700 species of cone snails in the world, and they are distributed in shallow water areas of tropical seas, mainly in the Indian Ocean and the Pacific Ocean. [0003] Cono snails can be divided into three categories according to their feeding habits: piscivorous, molluscivorous and vermivorous. Among them, the insectivorous cone snails are the most species, accounting for about 70% of all cone snail species, while the piscivorous cone snails account for the least amount of the total number of cone snails, but they are the most toxic. Caused by cone snails. [0004] Conotoxin (CTx) is a kind of active polypeptide derived from the...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C07K14/435A61K38/17A61P25/04A61P9/10A61P25/08
Inventor 徐安龙任政华覃梦颖王磊吴赟周亮陈翠玲戴清
Owner SUN YAT SEN UNIV
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