Preparation and application of conotoxin Lt7b of Conus littertus Linnaeus of South China sea

A conotoxin and signaling technology, applied in the direction of application, medical preparations containing active ingredients, non-central analgesics, etc.

Inactive Publication Date: 2012-08-08
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, conotoxins whose functions have been clarified at home and abroad only account for a small part of the entire toxin library, but their physiological functions have been clearly understood

Method used

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  • Preparation and application of conotoxin Lt7b of Conus littertus Linnaeus of South China sea
  • Preparation and application of conotoxin Lt7b of Conus littertus Linnaeus of South China sea
  • Preparation and application of conotoxin Lt7b of Conus littertus Linnaeus of South China sea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[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, and DNA ladder were purchased from TaKaRa Company; dNTP mix, pGEM-T Easy Vector Systems were purchased from Promega Company; PCR primers were synthesized by Invitrogen Company; other organic reagents were of domestic analytical grade and purchased from Guangzhou Chemical reagent factory.

[0038] Take live snails, break the snail shell with a 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 50-100mg tissue sample, use 1ml Homoge...

Embodiment 2

[0069] Example 2: Construction of fusion expression vector of signal toxin polypeptide Lt7b from South China Sea

[0070] 1) Plasmids and strains:

[0071] Escherichia coli (Escherichia coli) DH5α was purchased from Invitrogen and kept in our laboratory, its genotype is: DH5α: supE44 Δlac U169 hsdR17 recA1 endAl gyrA96 thi-1 rel A1; Escherichia coli expression host strain BL21(DE3) was purchased from Stratagene, and the genotype was: BL21(DE3): F-ompT hsdSB(r-B, m-B)dcm galλ(DE3).

[0072] 2) Reagents and other materials:

[0073] Restriction enzymes Kpn I and Not I, D12000DNAMarker were purchased from TaKaRa Company, T4DNA Ligase was purchased from Promega Company; Taq DNA polymerase, 10×PCR Buffer and dNTP were purchased from Dingguo Company; Gel Extraction Kit and Plasmid Miniprep Kit were purchased from OMEGA BIOTEK products; BCA TM Protein Assay Kit is a product of PIERCE; low molecular weight standard protein 14.4-108kD was purchased from KGI; EK enzyme was purchase...

Embodiment 3

[0101] Example 3: Efficient prokaryotic expression of the South China Sea signal conotoxin polypeptide gene

[0102] 1) Expression of fusion protein

[0103] The recombinant vector pTRX-Lt7.2 with correct sequencing was transformed into Escherichia coli BL21(DE3) to construct an engineering strain. 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 solution to a final concentration of 0.2%, and induced expression at 18°C ​​for 10 hours. After induction, centrifuge at 4°C and 10,000rpm for 10min, collect the bacteria, and resuspend with pre-cooled ultrasonic buffer (50mmol / L Tris, 500mmol / L NaCl, 20mmol / L imidazole, pH 7.0) at a ratio of 1:10...

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Abstract

The present invention relates to a preparation method and application of conotoxin gene Lt7.2 of Conus littertus Linnaeus of South China sea and polypeptide Lt7b encoded thereby. Through construction of a cDNA library, a conotoxin gene Lt7.2 is cloned by the invention from poison canal tissue of the Conus littertus Linnaeus of South China sea. The invention provides a preparation method of polypeptide. The conotoxin gene Lt7.2 and a vector pTRX are connected to get the recombinant expression vector pTRX-Lt7.2; the recombinant expression vector pTRX-Lt7.2 is transformed into host bacteria and expresses in the host bacteria stably and efficiently; and expression product recombinant fusion protein is separated and purified to obtain an objective protein polypeptide Lt7b. The polypeptide Lt7b provided by the invention has effects of neurotransmitter blocking transport and central analgesia and can be used for preparing tool medicine for the neurobiological research and analgesic drugs.

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 snails, commonly known as "cone snails", belong to Mollusca, Gastropoda, Prosobranchia and Neogastropoda in taxonomy. , Conidae, Conus. There are more than 700 species of cone snails existing on the earth, mainly distributed in warm tropical or subtropical seas, among which the Indo-Pacific region and the East Pacific-Western Atlantic region are the most numerous. Cone snails generally live on rocky, coral reefs, sandy and silty seabeds. A few species can inhabit deep water areas ranging from tens of meters to more than 200 meters. It inhabits under seaweed or in coral caves during the day or after low tide, and goes out to look for food at night. Spring and summer are its main breeding seasons. About 80 kinds of cone snails have been found in our country, mainly di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/63C12N15/70C12Q1/02A61K38/17A61P29/00
Inventor 徐安龙吴赟王磊任政华周茂军尤驭文朱晓燕强媛媛
Owner SUN YAT SEN UNIV
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