Cloning, recombinant expression, and characterization of a Rhipicephalus microplus carboxylesterase.

Gates Open Research Pub Date : 2024-08-19 eCollection Date: 2024-01-01 DOI:10.12688/gatesopenres.16245.1

Michel Labuschagne

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引用次数: 0

Abstract

Background: The Rhipicephalus microplus carboxylesterase (CBE) is involved in synthetic pyrethroid (SP) hydrolysis and historic evidence suggests that a non-synonymous mutation (Asp374Asn) in CBE is associated with increased resistance towards SP-based acaricides. Functional expression and characterization of the wild-type and mutant CBE is required to understand the impact of the mutation on SP-based resistance.

Methods: The R. microplus CBE gene was cloned and functionally expressed in Pichia pastoris following the removal of the native signal peptide. Site directed mutagenesis was used to introduce the Asp374Asn substitution.

Results: Functional expression, characterization, and purification of both wild-type and mutant R. microplus CBE proteins was achieved using affinity chromatography under native conditions.

Conclusions: This report provides the necessary information for the tick research community to produce recombinant tick derived CBE proteins and to characterize the recombinant proteins towards substrates of interest.

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微根头菌羧酸酯酶的克隆、重组表达和特性研究。

背景：微根头虫羧酸酯酶（CBE）参与合成拟除虫菊酯（SP）的水解，历史证据表明，CBE的非同义突变（Asp374Asn）与对基于SP的杀螨剂的抗性增加有关。为了了解突变对sp基抗性的影响，需要了解野生型和突变型CBE的功能表达和特征。方法：克隆微弧菌CBE基因，在毕赤酵母中去除其天然信号肽，进行功能表达。采用定点诱变技术引入Asp374Asn取代。结果：在自然条件下，利用亲和层析技术，实现了野生型和突变型R. microplus CBE蛋白的功能表达、鉴定和纯化。结论：本报告为蜱研究界生产重组蜱源CBE蛋白和对感兴趣的底物表征重组蛋白提供了必要的信息。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Gates Open Research Immunology and Microbiology-Immunology and Microbiology (miscellaneous)

CiteScore

3.60

自引率

0.00%

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