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2025年4月4日 星期五
农业生物技术学报  2020, Vol. 28 Issue (10): 1830-1836    DOI: 10.3969/j.issn.1674-7968.2020.10.011
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
捻转血矛线虫ABC转运蛋白的基因克隆、原核表达及纯化
赵学亮1,2, 王姝懿1, 孙柯1, 呼和巴特尔1, 王文龙1,*, 刘春霞3,*
1 内蒙古农业大学 兽医学院/农业农村部动物疾病临床诊疗技术重点实验室,呼和浩特 010018;
2 西北农林科技大学 动物医学院,杨凌 712100;
3 内蒙古农业大学 生命科学学院,呼和浩特 010018
Gene Cloning, Prokaryotic Expression and Purification of ABC Transporter in Haemonchus contortus
ZHAO Xue-Liang1,2, WANG Shu-Yi1, SUN Ke1, Huhe Bateer1, WANG Wen-Long1,*, LIU Chun-Xia3,*
1 College of Veterinary Medicine, Inner Mongolia Agricultural University/Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture and Rural Affairs, Hohhot 010018, China;
2 College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;
3 College of Life Science, Inner Mongolia Agricultural University, Hohhot 010018, China
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摘要 捻转血矛线虫(Haemonchus contortus)是绵羊(Ovis aries)、山羊(Capra hircus)等反刍动物重要的寄生虫之一,可造成养殖业的巨大经济损失。基于转录组研究,本课题组前期从捻转血矛线虫耐药虫株中鉴定获得耐药基因—ABC (ATP-binding cassette)转运蛋白基因(GenBank No. MT478136)。本研究拟分析ABC转运蛋白基因的生物信息学特征,建立ABC转运蛋白的可溶性诱导表达及纯化体系,为进一步探究ABC转运蛋白参与的耐药机制提供参考依据。首先利用在线软件对ABC进行蛋白质结构分析及抗原表位预测,结果显示,ABC分子式为C928H1502N270O265S7,属于不含跨膜区和信号肽的亲水性蛋白质,二级结构主要以延伸链为主;ABC转运蛋白的抗原性较高,含有较多的抗原表位。利用反转录PCR (reverse transcription PCR, RT-PCR)扩增ABC转运蛋白基因,经酶切连接构建重组质粒pET30a(+)-ABC,转化大肠杆菌(Escherichia coli) BL21后进行诱导表达条件的优化,以0.6 mmol/L异丙基硫代半乳糖苷(isopropyl β-D-thiogalactoside, IPTG)于37 ℃诱导7 h可重组表达1 mg/mL ABC转运蛋白。本研究成功诱导表达并纯化了捻转血矛线虫重组ABC转运蛋白,可为深入开展其潜在耐药机制及免疫原性研究工作提供基础资料。
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赵学亮
王姝懿
孙柯
呼和巴特尔
王文龙
刘春霞
关键词 捻转血矛线虫ABC (ATP-binding cassette)转运蛋白基因生物信息学分析原核表达蛋白纯化    
AbstractHaemonchus contortus is one of the most important parasites of ruminants such as Ovis aries and Capra hircus, and can bring huge economic losses to the breeding industry. In preliminary transcriptome study of present research group, the resistant gene ATP-binding cassette (ABC) transporter gene (GenBank No. MT478136) was identified from the resistant strain of Haemonchus contortus. The purpose of this study was to explore the bioinformatics character of the ABC transporter gene, to establish a system of soluble expression and purification for ABC transporter, and to lay the foundation for further exploration of ABC transporter-involved drug resistance mechanism. The online software was used to predict the protein structure and antigen epitope. The results showed that the molecular formula of ABC was C928H1502N270O265S7, which was a hydrophilic protein without transmembrane region and signal peptide. The secondary structure was mainly extended chains. The prediction of antigenic epitopes indicated that ABC transporter had many antigenic epitopes with high antigenicity. The ABC transporter gene was amplified by reverse transcription PCR (RT-PCR), and the recombinant plasmid pET30a(+)-ABC was constructed by restriction digestion. The plasmid was transformed into Escherichia coli BL21 to optimize inducing conditions. It was found that 1 mg/mL recombinant ABC transporter could be expressed with 0.6 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 37 ℃ for 7 h. In this study, recombinant ABC transporter was successfully expressed and purified in Haemonchus contortus, which could provide a reference for further mechanism study on potential drug resistance and immunogenicity.
Key wordsHaemonchus contortus    ATP-binding cassette (ABC) transporter gene    Bioinformatics analysis    Prokaryotic expression    Protein purification
收稿日期: 2020-03-11     
ZTFLH:  S855.9  
基金资助:内蒙古自治区科技计划项目(201702074); 国家自然科学基金(31760731)
通讯作者: * wwl.imau@163.com;lcx.imau@163.com   
引用本文:   
赵学亮, 王姝懿, 孙柯, 呼和巴特尔, 王文龙, 刘春霞. 捻转血矛线虫ABC转运蛋白的基因克隆、原核表达及纯化[J]. 农业生物技术学报, 2020, 28(10): 1830-1836.
ZHAO Xue-Liang, WANG Shu-Yi, SUN Ke, Huhe Bateer, WANG Wen-Long, LIU Chun-Xia. Gene Cloning, Prokaryotic Expression and Purification of ABC Transporter in Haemonchus contortus. 农业生物技术学报, 2020, 28(10): 1830-1836.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.10.011     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I10/1830
 
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