林氏扇头蜱几丁质酶基因RLCht1差异表达分析及酶活性检测

doi:10.16431/j.cnki.1671-7236.2024.11.031

摘要/Abstract

摘要： 【目的】蜱的蜕皮发育过程与几丁质合成和水解紧密相关，而几丁质的降解主要是通过几丁质酶来完成。试验旨在分析林氏扇头蜱(Rhipicephalus linnaei)不同时期及各组织中几丁质酶RLCht1基因序列表达差异、体外异源表达RLCht1蛋白并验证其几丁质酶活性，为开发安全高效的生物杀蜱剂提供新型靶标思路。【方法】收集不同吸血状态的各时期蜱及雌性饱血成蜱的不同组织样本用于基因差异表达分析，基于血红扇头蜱合神经节转录组中筛选出的推测的几丁质酶(putative chitinase)序列，在林氏扇头蜱中进行扩增并构建重组原核质粒，使用大肠杆菌系统表达林氏扇头蜱几丁质酶蛋白RLCht1，进行包涵体处理后使用镍柱纯化蛋白，预测RLCht1蛋白结构域；通过与3种不同荧光底物发生催化反应，检测纯化的几丁质酶RLCht1的催化活性大小及作用方式。【结果】 RLCht1基因在不同饱血状态下的各个时期及饱血雌成蜱的不同组织中均有表达，饱血若蜱阶段表达量极显著高于卵期(P＜0.01),显著高于其他时期(P＜0.05),并在饱血雌性成蜱中肠内有较高的表达量；构建了重组质粒RLCht1-pET-28a并成功表达RLCht1蛋白，经包涵体处理后在150 mmol/L咪唑洗脱下得到纯化的RLCht1蛋白，蛋白预测结果显示，RLCht1属于GH18家族几丁质酶；荧光底物酶活性检测表明，RLCht1包涵体溶解液、复性未纯化的原核蛋白、复性纯化浓缩后原核蛋白分别具有0.078与0.350 U/mL的几丁质外切酶活性及0.034 U/mL β-N-乙酰氨基葡萄糖苷酶活性。【结论】 RLCht1基因与若蜱至成蜱阶段的蜕皮活动相关，影响与免疫、消化、体液调节有关的生命活动过程。本研究在林氏扇头蜱中成功表达几丁质酶蛋白，证明了纯化的RLCht1蛋白具有几丁质催化功能，为林氏扇头蜱几丁质酶RLCht1蛋白在今后杀蜱剂开发中的应用提供了理论基础。

关键词: 林氏扇头蜱; 酶活性检测; 表达谱; 原核表达; 包涵体

Abstract: 【Objective】 The molting process of ticks was closely related to chitin synthesis and hydrolysis,and chitin degradation is mainly done by chitinase,analysis of different periods and tissue in the tick (Rhipicephalus linnaei) chitinase RLCht1 sequence expression profile,in vitro heterologous expression protein and verify the activity of chitinase,for the tick and other vector biological control of reference were carried out,so as to provide a new target for developing safe and efficient biological tick. 【Method】 Different tissue samples of different period ticks and female blood-fed ticks were collected for differential expression analysis.Based on the putative chitinase sequence screened from the transcriptome of Rhipicephalus sanguineus,the recombinant prokaryotic plasmid was amplified and constructed in Rhipicephalus linnaei,and the E.coli system was used to express the chitinase protein RLCht1.After the inclusion body treatment,the protein was purified by nickel column and the RLCht1 protein domain was predicted. After purification using a nickel column,RLCht1 protein domain structure was predicted.The catalytic activity size and mode of action of purified chitinase RLCht1 were tested by a catalytic reaction with three different fluorescent substrates. 【Result】 The RLCht1 gene was expressed at different stages of satiety and in different tissues of satiated female adult ticks.The expression of RLCht1 gene in the satiated tick stage was extremely significantly higher than that in the egg stage (P＜0.01) and other stages (P＜0.05),and had a higher expression in the midgut of satiated female adult ticks.The recombinant plasmid RLCht1-pET-28a was constructed and the RLCht1 protein was successfully expressed.After inclusion body treatment,the purified RLCht1 protein was obtained by elution at a concentration of 150 mmol/L imidazole.The protein prediction results showed that RLCht1 belonged to the GH18 family chitinase.Fluorescent substrate enzyme activity detection showed that RLCht1 inclusion body dissolution solution,refolded unpurified prokaryotic protein,and refolded purified concentrated prokaryotic protein had chitin exonuclease activity of 0.078 and 0.350 U/mL,and β-N-acetylglucosaminidase activity of 0.034 U/mL,respectively. 【Conclusion】 RLCht1 might be related to the activity of ticks,affect life activities related to immunity,digestion and humoral regulation.The study successfully expressed chitinase protein for the first time in the Rhipicephalus linnaei,demonstrating that the purified RLCht1 protein had chitin catalytic function and provided a theoretical basis for the application of chitinase RLCht1 protein in the development of ticks in the future.

Key words: Rhipicephalus linnaei; enzyme activity assay; expression spectrum; prokaryotic expression; inclusion body

中图分类号:

S852.74⁺6

李尧, 赵培真, 谢子芳, 彭维祺, 陈杰, 赵建国, 管庆丰. 林氏扇头蜱几丁质酶基因RLCht1差异表达分析及酶活性检测[J]. 中国畜牧兽医, 2024, 51(11): 4964-4973.

LI Yao, ZHAO Peizhen, XIE Zifang, PENG Weiqi, CHEN Jie, ZHAO Jianguo, GUAN Qingfeng. Differential Expression Analysis of RLCht1 Gene in Rhipicephalus linnaei and Detection of Enzyme Activity[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(11): 4964-4973.

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