Modified product of reconstructed sea-anemone neurotoxin rhk2a, modifying method and application thereof

A neurotoxin and modification method technology, applied in the field of modified products of recombinant sea anemone neurotoxin rhk2a, can solve problems such as modification, no recombinant sea anemone peptide toxoid, etc., achieve good uniformity, simple and easy modification method, The effect of high activity recovery rate

Inactive Publication Date: 2009-09-23
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, there are still no technical reports on modifying the recombinant sea anemone peptide toxoid rhk2a to reduce its toxicity. Therefore, how to obtain a feasible technical solution to obtain a low-toxicity and stable recombinant sea anemone neurotoxin through rhk2a modification, improve The pharmacokinetics of the protein to improve the stability in vivo and in vitro and the convenience of administration, so that rhk2a can play an important clinical application, has always been the focus and urgent point of research

Method used

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  • Modified product of reconstructed sea-anemone neurotoxin rhk2a, modifying method and application thereof
  • Modified product of reconstructed sea-anemone neurotoxin rhk2a, modifying method and application thereof
  • Modified product of reconstructed sea-anemone neurotoxin rhk2a, modifying method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The condition determination of embodiment 1 modification method

[0041] 1. Selection of reaction pH value: Prepare 1ml of rhk2a solution of 1 mg / ml with 0.02M acetic acid buffer solution with pH value of 3.5, 4.0, 5.0, 6.0, respectively, and add monomethoxy polyethoxylate with a molar ratio of 1:5 respectively. Ethylene glycol propionaldehyde 5000 (mPEG-ALD-5000), stirred at room temperature for 1 hour and mixed, then added 0.02ml of 1M sodium cyanoborohydride and stirred for reaction. After reacting for 8 hours, the modification rate of the reactant was measured. The results are shown in Table 1 As shown, the polyethylene glycol-modified sea anemone neurotoxin rhk2a can be obtained in buffer solutions with pH values ​​of 3.5, 4.0, 5.0, and 6.0, and the modification rate of the buffer solution with a pH value of 4.0 is the highest.

[0042] Table 1

[0043] Buffer pH

3.5

4

5

6

[0044] Modification rate (%)

53.8

...

Embodiment 2

[0057] Example 2 Isolation and identification of modified products

[0058] Use 0.02M acetic acid buffer solution with pH value 4.0 to prepare 2ml of 1mg / ml rhk2a solution, add 10mg of monomethoxy polyethylene glycol propionaldehyde 5000 (mPEG-ALD-5000) with a molar ratio of 1:5 and stir at room temperature for 1 After mixing for 1 hour, add 0.08ml of 1M sodium cyanoborohydride and stir for 12 hours to obtain reaction solution 1;

[0059] Use 0.02M acetic acid buffer solution with pH value 4.0 to prepare 2ml of 1mg / ml rhk2a solution, add 10mg of monomethoxypolyethylene glycol butyraldehyde 10000 (mPEG-ButyrALD-10000) with a molar ratio of 1:10 and stir at room temperature for 1 Mix for 1 hour, add 0.08ml of 1M sodium cyanoborohydride and stir for 12 hours to obtain reaction solution 2;

[0060] 2ml of 1mg / ml rhk2a solution was prepared by using 0.02M acetic acid buffer solution with a pH value of 4.0, and 15mg of activated polyethylene glycol propionaldehyde 20000 (mPEG-ALD-2...

Embodiment 3

[0072] Example 3 RP-HPLC identification of modified purified products

[0073] The RP-HPLC method is a chromatographic separation mode based on the hydrophobic interaction between the solute, polar mobile phase and non-polar groups on the surface of the stationary phase. After being modified by hydrophilic polyethylene glycol, the number, type and distribution of hydrophobic groups on the surface of the protein will change, so the hydrophobic interaction with the non-polar groups on the surface of the stationary phase will be different, thereby achieving separation.

[0074] The purified and separated polyethylene glycol-modified sea anemone neurotoxin rhk2a freeze-dried product was dissolved in a small amount of ultrapure water, and then analyzed and identified by PR-HPLC. The analysis conditions are as follows:

[0075] 1) Chromatographic column: waters Symmetry300C18, specification: 250mm×4.6mm

[0076] 2) Mobile phase:

[0077] A: 0.1% trifluoroacetic acid (w / v)

[007...

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Abstract

The invention discloses a modified product of reconstructed sea-anemone neurotoxin rhk2a, a modifying method and an application thereof. The modified product of the reconstructed sea-anemone neurotoxin rhk2a is prepared by connecting a polyglycol chain with single site specifically at an amino N end of the reconstructed sea-anemone neurotoxin rhk2a. The modified product has good equality, simple separation and purification technologies and high activated coefficient of recovery, and polyglycol site-modified sea-anemone neurotoxin rhk2a has extremely good application prospects in an aspect of treating congestive heart failure.

Description

technical field [0001] The invention belongs to the field of biotechnology pharmaceutical preparations, and specifically relates to a modified product of recombinant sea anemone neurotoxin rhk2a, a modification method and application thereof. Background technique [0002] In recent years, with the development of biotechnology, various active proteins and cytokines have been developed into drugs for clinical application. However, it is an urgent problem to make protein drugs into suitable dosage forms to protect them from external environmental damage, prolong drug half-life, and reduce toxic and side effects. Among them, the study of protein PEGylation has become one of the hot spots that scholars at home and abroad pay attention to. This is a new technology developed in the past 30 years to improve the pharmacokinetic properties of protein drugs in vivo. It bonds activated polyethylene glycol molecules to the surface of protein molecules, affects the spatial structure of p...

Claims

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Application Information

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IPC IPC(8): C07K17/08
Inventor 杨帆潘育方黄慧雷彤
Owner GUANGDONG PHARMA UNIV
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