基于转录组测序的冈田绕眼果蝇aoattacin基因筛选及在昆虫细胞中表达、抑菌活性检测

doi:10.13560/j.cnki.biotech.bull.1985.2019-0157

生物技术通报 ›› 2019, Vol. 35 ›› Issue (9): 118-124.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0157

基于转录组测序的冈田绕眼果蝇aoattacin基因筛选及在昆虫细胞中表达、抑菌活性检测

蔡娟¹, 刘流¹, 王灵军¹, 曹建平², 郑明辉^1,2, 刘晖¹

1. 遵义医科大学,遵义 563003;
2. 中国疾病预防控制中心寄生虫病预防控制所,上海 200025

收稿日期:2019-02-27 出版日期:2019-09-26 发布日期:2019-09-16
作者简介:蔡娟,女,硕士研究生,研究方向：寄生虫感染与免疫：E-mail：446487619@qq.com
基金资助:
国家自然科学基金项目（81560336,81760373）,贵州省科技厅科技基金（黔科合基础[2016]1168,黔科合J字[2010]2160）,遵义医学院博士启动基金（F-795）,卫生部寄生虫病原与媒介生物学重点实验室开放课题（WSBKFKT-201609）

Screening aoattacin Gene from Amiota okadai Based on Transcriptome Sequencing,Expression of It in Insect Cells and Antibacterial Activity Identification

CAI Juan¹, LIU Liu¹, WANG Ling-jun¹, CAO Jian-ping², ZHENG Ming-hui^1,2, LIU Hui¹

1. Zunyi Medical University,Zunyi 563003;
2. National Institute of Parasitic Diseases Chinese Center For Disease Control And Prevention,Shanghai 200025

Received:2019-02-27 Published:2019-09-26 Online:2019-09-16

摘要/Abstract

摘要： 通过分析冈田绕眼果蝇转录组数据得到抗菌肽aoattacin基因,在昆虫细胞Sf9中表达并检测其抑菌活性。通过转录组分析得到抗菌肽aoattacin基因,全基因合成其序列,构建重组pFast-bac1-aoattacin载体,通过转染DH10Bac细胞获得重组杆状病毒,转染昆虫Sf9细胞,以获得P1代病毒和通过扩增获得P2代病毒,并检测基因在Sf9细胞里的表达。通过SDS-PAGE和Western Blot检测蛋白表达情况,通过最小抑菌浓度测定表达产物的抑菌活性。结果表明通过筛选转录组数据得到一个高表达aoattacin基因,在昆虫细胞Sf9中成功表达,抑菌实验显示抗菌肽Aoattacin 对受试革兰氏阳性及阴性菌等多种细菌具有抑制作用,或为一种新型广谱抗菌肽。

关键词: 冈田绕眼果蝇, 转录组, 抗菌肽, 真核表达, 抑菌活性

Abstract: By analyzing the transcriptome data of Amiota okadai,an attacin homologous unigene named aoattacin was obtained,which was expressed in insect cell Sf9 and its antibacterial activity was detected. The antibacterial peptide aoattacin gene was obtained by transcriptome analysis. The antibacterial peptide aoattacin gene was obtained by transcriptome analysis,and the sequence of the whole gene was synthesized to construct the recombinant pFast-bac1-aoattacin vector. The recombinant vector was transfected into DH10Bac cells to obtain recombinant baculovirus,which was transfected into insect Sf9 cells. To obtain the P1 virus and obtain the P2 virus by amplification,and detect the expression of the gene in Sf9 cells. Protein expression was detected by SDS-PAGE and Western Blot,and the antibacterial activity of the expressed product was determined by the minimum inhibitory concentration. The results showed that a high-expressed aoattacin gene screening from transcriptome data was successfully expressed in insect cell Sf9,and Aoattacin demonstrated antibacterial activity against many kinds of bacteria in experiments,such as both Gram-positive bacteria and Gram-negative bacteria,which may be a new broad-spectrum antimicrobial peptide.

Key words: Amiota okadai, transcriptome, antimicrobial peptide, Eukaryotic expression, antibacterial activity

蔡娟, 刘流, 王灵军, 曹建平, 郑明辉, 刘晖. 基于转录组测序的冈田绕眼果蝇aoattacin基因筛选及在昆虫细胞中表达、抑菌活性检测[J]. 生物技术通报, 2019, 35(9): 118-124.

CAI Juan, LIU Liu, WANG Ling-jun, CAO Jian-ping, ZHENG Ming-hui, LIU Hui. Screening aoattacin Gene from Amiota okadai Based on Transcriptome Sequencing,Expression of It in Insect Cells and Antibacterial Activity Identification[J]. Biotechnology Bulletin, 2019, 35(9): 118-124.

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基于转录组测序的冈田绕眼果蝇aoattacin基因筛选及在昆虫细胞中表达、抑菌活性检测

Screening aoattacin Gene from Amiota okadai Based on Transcriptome Sequencing,Expression of It in Insect Cells and Antibacterial Activity Identification

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