布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.08.007

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (8): 2746-2753.doi: 10.16431/j.cnki.1671-7236.2021.08.007

布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析

杨江流¹, 乔军伟¹, 宋青山¹, 贾芳^1,2

1. 河套学院医学系, 巴彦淖尔 015000;
2. 宁夏大学生命科学学院, 西部特色生物资源保护与利用教育部重点实验室, 银川 750021

修回日期:2021-02-13 出版日期:2021-08-20 发布日期:2021-08-19
通讯作者: 贾芳 E-mail:jiafang198011@163.com
作者简介:杨江流(1984-),男,宁夏隆德人,硕士,讲师,研究方向:动物病原微生物,E-mail:nptt2005@163.com
基金资助:
内蒙古自治区高等学校科学研究项目（NJZY20226）

Prokaryotic Expression and Bioinformatics Analysis of Virulence Gene BvfA of Brucella S19

YANG Jiangliu¹, QIAO Junwei¹, SONG Qingshan¹, JIA Fang^1,2

1. Department of Medicine, Hetao College, Bayannur 015000, China;
2. Key Laboratory of the Ministry of Education for the Conservation and Utilization of Special Biological Resources of Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China

Revised:2021-02-13 Online:2021-08-20 Published:2021-08-19

摘要/Abstract

摘要： 试验旨在对布鲁氏菌S19毒力因子A（BvfA）基因进行克隆及原核表达，并对其编码的BvfA蛋白结构进行生物信息学分析。登录GenBank下载布鲁氏菌S19的1号染色体（登录号：CP030751.1），根据文献报道的布鲁氏菌S19 BvfA基因的位置和BvfA蛋白的分子质量，在NCBI的ORF Finder中预测编码BvfA蛋白的开放阅读框（ORF），根据ORF预测的BvfA基因设计扩增BvfA基因的引物，克隆该序列并连接到表达载体pET-32a（+）上，构建重组质粒pET-32a（+）-BvfA，并诱导其在大肠杆菌BL21（Ril）感受态细胞中表达，表达产物经SDS-PAGE和Western blotting验证，并用生物信息学方法对BvfA蛋白结构进行在线预测。结果显示，BvfA基因ORF全长为336 bp，表达纯化得到分子质量为11 ku的分泌型BvfA蛋白；生物信息学分析显示，BvfA蛋白属于不稳定的亲水性蛋白，无跨膜结构，第1-28位氨基酸处存在信号肽区域，BvfA蛋白55.6%定位于细胞外，有12个可能的磷酸化位点，无O-糖基化位点，BvfA蛋白二级结构主要由α-螺旋和无规则卷曲组成。以上结果可为后续研究BvfA蛋白在布鲁氏菌中的致病机制及寻找布鲁氏菌病新的治疗靶点提供一定的参考。

关键词: 布鲁氏菌; BvfA; 原核表达; 生物信息学分析

Abstract: The objective of this study was to clone and express virulence gene BvfA of Brucella (B. abortus) S19, and analyzed the bioinformatics of its expressed protein.In the tests, chromosome 1(accession No.:CCP030751.1)of B.arbortus S19 was downloaded from GenBank.According to the reported position of BvfA gene and molecular weight of BvfA protein in B.arbortus S19, the Open Read Frame (ORF) of BvfA proteins in NCBI's ORF Finder was predicted.The obtained BvfA gene was ligated into prokaryotic expression vector pET-32a(+) to construct the recombinant plasmid pET-32a(+)-BvfA, and expressed in Escherichia coli BL21(Ril).The expressed products were analysed by SDS-PAGE and Western blotting, the structure of BvfA protein was predicted by bioinformatics methods.The results showed that the full-length ORF of BvfA gene was 336 bp, and the expressed fusion BvfA protein was about 11 ku.Bioinformatics analysis showed that BvfA was an unstable hydrophilic protein with no transmembrane structure and its signal peptide region was 1-28 amino acid, BvfA protein mainly located in the extracellular protein (55.6%).There were 12 potential phosphorylation sites, and no O-glycosylation sites.BvfA protein secondary structure was mainly composed of alpha helix and random coil.The results of this study provided some references for the further study of the pathogenic mechanism of BvfA protein in Brucella and search for new therapeutic targets for brucellosis.

Key words: Brucella; BvfA; prokaryotic expression; bioinformatics analysis

中图分类号:

S852.61⁺4

杨江流, 乔军伟, 宋青山, 贾芳. 布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(8): 2746-2753.

YANG Jiangliu, QIAO Junwei, SONG Qingshan, JIA Fang. Prokaryotic Expression and Bioinformatics Analysis of Virulence Gene BvfA of Brucella S19[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2746-2753.

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布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析

Prokaryotic Expression and Bioinformatics Analysis of Virulence Gene BvfA of Brucella S19

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