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Venin-derived cytotoxin CTXn for drug rehabilitation and purification method and application thereof

A cytotoxin, purification method technology, applied in the field of biopharmaceuticals

Inactive Publication Date: 2010-06-02
广州医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether there is CTXn in Zhoushan cobra venom in other parts of mainland China, and whether it has the function of detoxification has not been reported so far

Method used

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  • Venin-derived cytotoxin CTXn for drug rehabilitation and purification method and application thereof
  • Venin-derived cytotoxin CTXn for drug rehabilitation and purification method and application thereof
  • Venin-derived cytotoxin CTXn for drug rehabilitation and purification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0040] Specific Example 1: Screening for morphine-addicted rats (morphine-induced conditioned place preference model, CPP)

[0041] 1) Collect Zhoushan cobra venom samples outside;

[0042] 2) Carry out the separation and purification of CTXn to the aforementioned Zhoushan cobra venom samples,

[0043] (1) First perform gel filtration: use Sephacryl S-100HR filler, filter Zhoushan cobra venom samples through the AKTAexplorer 100 rapid purification process development system for molecular sieve filtration to obtain I, II and III toxic components, which are pre-treated with G-50 respectively Pack HiPrep 26 / 10 Desalting desalting column, freeze-dried, and set aside;

[0044] (2) Through preliminary screening, select component III for the next step of "ion exchange";

[0045] (3) Ion exchange: Using CM Sepharose FF filler, component III was ion exchanged through the AKTA explorer 100 rapid purification process development system to obtain a single toxic polypeptide CTXn( figure...

specific Embodiment 2

[0053] Specific Example 2: The method of administering CTXn to morphine-addicted rats

[0054] The morphine-addicted rats were randomly divided into 5 groups: 3 dose groups of CTXn (2.0mg / kg, 1.0mg / kg, 0.5m / kg) were injected intraperitoneally with snake venom at 8.00 am and 0.9% intraperitoneally at 16.00 pm for 8 consecutive days; NS, The morphine addicted control group was subcutaneously injected with 0.9% NS at the same time, the training procedure and the establishment of the conditional place preference model were tested. Record the rat's body weight, measure CPP, observe its mental state and activity every day and record them. On day d18, 5 mg / kg naloxone was injected intraperitoneally to observe the withdrawal symptoms and score them. Withdrawal for 4 days, d22 test.

specific Embodiment 3

[0055] Specific Example 3: Effects of Different Doses of CTXn on Morphine-Induced Conditioned Place Preference (CPP)

[0056] There was no significant difference in the time of staying in the white box before the rats of each group were given morphine (P>0.05), and the time of the rats staying in the white box was significantly prolonged after 8 consecutive days of morphine training in the morphine group and each dose group of CTXn. Compared with the former group, there was a significant difference (P<0.01), and compared with the 0.9% NS group, there was also a significant difference (P<0.01). After the rats produced a stable CPP to morphine, they were given CTXn 2mg / kg, 1mg / kg, and 0.5mg / kg doses for 8 days each, and on d10 days, the CTXn 2mg / kg and CTXn 1mg / kg dose groups were compared with the morphine control group and the given doses. There was a significant difference before CTXn (P<0.01); after being treated with CTXn for 8 consecutive days, the phenomenon of preference...

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Abstract

The invention relates to venin-derived cytotoxin CTXn for drug rehabilitation and a purification method and application thereof, which is characterized in that: 1. the molecular weight of the CTXn is 6697.429Da; 2. the amino acid sequence of the CTXn is LKCNQLIPPF YKTCAAGKNLCYKMFMVAAP KVPVKRGCID VCPKSSLLVK YVCCNTDRCN. A preparation method comprises the following steps of: collecting a Zhoushan cobratoxin specimen outside; carrying out CTXn separation and purification for the Zhoushan cobratoxin specimen, firstly carrying out gel filtration, secondly selecting a component III for the next step of ion exchange through primary screening, and thirdly carrying out ion exchange which adopts a rapid purification process development system, obtaining single toxic polypeptide CTXn, desalting with a G-50 prepacked column, and freezing to dryness for standby; and measuring the molecular weight of the CTXn, the amino acid sequence thereof and the amino acid molecular weight. The venin-derived cytotoxin CTXn can eliminate the drug thirsting behaviors and withdrawal symptoms of addictive rats for morphine, has the characteristics of remarkable drug effect and small toxic or side effect, is favorable for large-scale production, and has great social meaning and economic value for preventing drug harms.

Description

technical field [0001] The invention relates to a cytotoxin CTXn derived from snake venom for detoxification and its purification method and application. The main use is a cytotoxin derived from the poison of Zhoushan cobra (Naja atra Cantor), which is suitable for the new pharmaceutical application of substituting the specific drug for opioid detoxification. It belongs to the technical field of biopharmaceuticals. Background technique [0002] At present, drug-abuse is a prominent social and medical problem at home and abroad. Since the 1990s, the drug problem in our country has become increasingly serious, and the number of drug abusers has increased dramatically. At present, the main drugs abused in our country are opium, morphine, heroin, marijuana, cocaine, amphetamine, etc. There are also some synthetic drugs, including methamphetamine and ecstasy. Drug abuse can seriously damage physical and mental health. Once addicted to drugs, it will lead to memory loss, severe...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/20C07K1/18A61K38/17A61P25/30
Inventor 孔天翰刘先国仲维高信文君董伟华崔跃黄绍崔超伟唐娅
Owner 广州医学院
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