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Preparation and application of conotoxin striatus S21a in South China Sea

A technology for conotoxin and cono snail, which is applied in the preparation and application field of conotoxin S21a in the South China Sea, and can solve the problems that the subunit composition cannot be clearly elucidated and the like.

Inactive Publication Date: 2011-08-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The subunit composition of these nAChRs has not been clearly elucidated due to the lack of selective ligands

Method used

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  • Preparation and application of conotoxin striatus S21a in South China Sea
  • Preparation and application of conotoxin striatus S21a in South China Sea
  • Preparation and application of conotoxin striatus S21a in South China Sea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0037] The extraction of total RNA refers to the method of Gibcol BRL company LS reagent instructions were carried out. Taq DNA polymerase, 10×PCRBuffer and dNTP were purchased from Dingguo Company; PCR primers were synthesized by Invitrogen Company; other organic reagents were domestic analytical reagents and were 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 50-100mg tissue sample, use 0.75ml Homogenize the LS reagent and let stand at 15-30°C for 5 minutes. Then add 0.2m...

Embodiment 2

[0066] Example 2: Construction of recombinant expression vector pTRX-S21.1

[0067] 1) Plasmids and strains:

[0068] Escherichia coli (Escherichia coli) DH5α was purchased from Invitrogen and kept by our laboratory, and its genotype is: DH5α: supE44Δlac U169 ( 80lacZAM15) hsdR17recA1endA1gyrA96thi-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).

[0069] 2) Reagents and other materials:

[0070] Restriction enzymes Kpn I, Not I and T4 DNA ligase were purchased from TaKaRa Company; Taq DNA polymerase, 10×PCRBuffer 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 is a product of PIERCE; Trans2K DNA Marker was purchased from TransGen; low molecular weight standard proteins 14,400-108,000kD and 14,300-97,200kD were purchased from KGI and TaKaRa respectively...

Embodiment 3

[0092] Example 3. Efficient prokaryotic expression of conotoxin polypeptide S21a from the South China Sea of ​​China

[0093] 1) Expression of recombinant fusion protein

[0094] The recombinant expression vector pTRX-S21.1 with correct sequencing was transformed into Escherichia coli BL21(DE3) to construct an engineering strain. Pick a single colony of the engineering strain and inoculate it in the Amp+LB liquid enriched medium, and culture it with shaking at 37°C overnight as the seed bacteria. Take the seed bacteria and inoculate them in fresh Amp+LB rich medium at a volume ratio of 1:100, amplify the culture with vigorous shaking at 37°C until the OD600 is about 0.6, add IPTG to a final concentration of 0.1mmol / L, and add 20% glucose to The final concentration is 0.2%, and the expression is induced for 12 hours at 18°C. After induction, centrifuge at 4°C and 10,000rpm for 10min to harvest the bacteria. Wash the bacteria once with pre-cooled PBS buffer (pH7.4), centrifuge...

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Abstract

The invention relates to a preparation method and an application of conotoxin striatus gene S21.1 in the South China Sea and the coded polypeptide S21a of the conotoxin striatus gene S21.1. According to the invention, a cDNA (complementary deoxyribonucleic acid) library is constructed, and the conotoxin striatus gene S21.1 is obtained by cloning in a tube of conus striatus in the South China Sea.The invention provides a preparation method of the polypeptide, which comprises the following steps: connecting the conotoxin gene S21.1 and a vector pTRX to obtain a recombinant expression vector pTRX-S21.1; transforming the recombinant expression vector pTRX-S21.1 into host bacteria and culturing and expressing in the host bacteria; and separating and purifying the recombinant fusion protein ofthe expression product to obtain the target protein, namely the polypeptide S21a. The polypeptide S21a provided by the invention has the functions of blocking neurotransmitter transfer and easing pains, and can be used for preparing tool medicines and analgesic medicines in neurobiology research.

Description

technical field [0001] The present invention relates to a polypeptide and its coding gene sequence, its preparation method and application. In particular, it relates to a conotoxin polypeptide and its coding gene sequence, its preparation method and application. Background technique [0002] Cone snail (cone snail) is a carnivorous animal that lives 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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N15/70C07K14/435A61K38/17A61P25/04A61P21/00
Inventor 徐安龙强媛媛王磊任政华吴赟覃梦颖
Owner SUN YAT SEN UNIV
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