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Activity combination screening method of botulinum neurotoxin proteins with different subtypes

A botulinum neurotoxin and screening method technology, applied in the field of biotechnology application research, to achieve the effect of improving hydrolysis efficiency

Inactive Publication Date: 2017-01-04
THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for screening the activity combination of different subtypes of botulinum neurotoxin proteins that may reduce or even eliminate the level of immune resistance, aiming to solve the problem that existing BoNTs therapeutic drugs are prone to immune resistance

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  • Activity combination screening method of botulinum neurotoxin proteins with different subtypes
  • Activity combination screening method of botulinum neurotoxin proteins with different subtypes

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Hydrolysis efficiency of a single botulinum neurotoxin light chain protease on endogenous natural substrates

[0040] First, construct recombinant vectors containing each subtype of botulinum neurotoxin light chain protease genes, express the several botulinum neurotoxin light chain proteases in Escherichia coli in the form of His tag fusion protein, and use Ni-NTA agrose( Invitrogen) affinity media separation and purification to obtain a fusion protein with higher purity and concentration;

[0041] Second, culture Neuro-2A cells (ATCC) according to conventional cell culture methods and following the supplier's recommendations;

[0042] Finally, mix a certain concentration of a single fusion protein with a certain volume of Neuro-2A cell lysate in a 10 μl reaction system (10mM Tris–HCl pH 7.6 and 20mM NaCl), place it in a 37°C constant temperature incubator for 20min, and then add SDS-PAGE sample buffer terminates the reaction.

[0043] The data obtained in ...

Embodiment 2

[0044] Example 2 The hydrolysis efficiency of endogenous natural substrates by combinations of different botulinum neurotoxin light chain proteases

[0045] In order to further analyze whether different combinations of botulinum neurotoxin light chain proteases can affect the hydrolysis efficiency of each light chain protease to its specific endogenous natural substrate, two different botulinum neurotoxin light chain proteases were combined with certain The volume of Neuro-2A cell lysate was mixed in 10 μl reaction system, and the experimental operation in Example 1 was repeated. The combination of botulinum neurotoxin light chain protease follows the following principles: LCs (such as LC / A and LC / E) that specifically hydrolyze SNAP-25 and LCs that specifically hydrolyze VAMP-2 (such as LC / B, LC / D, Combination between LC / F and LC / TeNT), that is, the two LCs in the combination hydrolyze different substrates.

[0046] The results are shown in Table 1. Table 1 shows that the co...

Embodiment 3

[0047] Example 3 The combination of LC / A&LC / B can significantly improve the hydrolysis efficiency of LC / A or LC / B to its specific endogenous natural substrate

[0048] The analysis of Examples 1 and 2 found that under the same standard conditions (i.e. the amount of LC required when 50% of the endogenous natural substrate was hydrolyzed), separate LC / A and LC / B Respectively >40000nM and 0.5nM were required to hydrolyze 50% of the corresponding substrate, but in the combination of the two LCs, this value was reduced to 800nM and 0.007nM, respectively, as shown in Table 1, figure 1 A and figure 1 As shown in B, figure 1 A represents LC / A alone and its hydrolytic activity on endogenous natural substrates exhibited in combinations LC / A&LC / B and LC / A&LC / F, figure 1 B represents the hydrolysis activity of LC / B alone and its combination LC / A&LC / B to endogenous natural substrates. It can be seen from the figure that the activities of LC / A and LC / B were increased by >50 times and ab...

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Abstract

The invention is applicable to the field of application research of biotechnology and provides an activity combination screening method of botulinum neurotoxin proteins with different subtypes. The activity combination screening method comprises the following steps: purifying to obtain recombinant protein of each subtype of botulinum neurotoxin light-chain proteinase and LC / TeNT (Light Chain-Tetanus Neurotoxin); detecting hydrolytic activity of the recombinant protein of each subtype of botulinum neurotoxin light-chain proteinase and the LC / TeNT to a specific recombinant substrate, and obtaining hydrolytic activity data of the specific recombinant substrate; detecting the hydrolytic activity of the recombinant protein of each subtype of botulinum neurotoxin light-chain proteinase and the LC / TeNT, and combined recombinant protein to a specific endogenous natural substrate, and obtaining hydrolytic activity data of the specific endogenous natural substrate; classifying, comparing and analyzing the hydrolytic activity data of the specific recombinant substrate and the hydrolytic activity data of the specific endogenous natural substrate; screening activity combination of the recombinant protein of each subtype of the botulinum neurotoxin light-chain proteinase.

Description

technical field [0001] The invention belongs to the field of biotechnology application research, and in particular relates to a method for screening activity combinations of different subtypes of botulinum neurotoxin proteins. Background technique [0002] Clostridial Neurotoxins (CNTs) are one of the strongest protein toxins that cause human poisoning, among which Botulinum Neurotoxins (BoNTs) cause muscle flaccid paralysis, Tetanus Neurotoxin (TeNT) causing spastic paralysis. The total length of CNTs is 150kDa. Overall, it can be divided into the N-terminal active region, namely the light chain (LC), which is about 50kDa, and the C-terminal receptor recognition and binding region, namely the heavy chain (heavy chain, HC). , about 100kDa, and the heavy chain can be further divided into two subregions, the light chain transport region (HCT) and the receptor binding region (HCR). For BoNTs, seven different serotypes (named in turn from BoNT-A to BoNT-G) have been isolated a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/50G01N33/68C12Q1/37
Inventor 陈声郭九标
Owner THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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