鼠伤寒沙门氏菌细菌铁蛋白(Bfr)的生物信息学分析及原核表达载体构建  

Bioinformatics analysis of bacterial ferritin(Bfr)from Salmonella typhimurium and construction of prokaryotic expression vector

作　　者：张伟[1,2] 史超[1,2] 于海涛 霍明凯 魏春燕[1,2] 黄炜涵 周霞 王震 张辉[1,2] ZHANG Wei;SHI Chao;YU Haitao;HUO Mingkai;WEI Chunyan;HUANG Weihan;ZHOU Xia;WANG Zhen;ZHANG Hui(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Key Laboratory of Animal Disease Prevention and Control,Xinjiang Production and Construction Corps,Shihezi 832003,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]新疆生产建设兵团动物疾病防控重点实验室,新疆石河子832003

出　　处：《黑龙江畜牧兽医》2024年第7期81-89,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：新疆生产建设兵团重点领域科技攻关项目(2021AB012)。

摘　　要：为了获得高纯度的鼠伤寒沙门氏菌细菌铁蛋白(Bfr),试验利用生物信息学软件对Bfr的结构、翻译后修饰和抗原表位等进行预测分析;通过PCR扩增鼠伤寒沙门氏菌Bfr基因,将其与pET-32a(+)载体连接并转化至大肠杆菌BL21(DE3)感受态细胞中,通过SDS-PAGE分析和Western-blot检测表达及纯化后的目的蛋白。结果表明:Bfr由476个氨基酸组成,分子式为C_(1440)H_(2406)N_(476)O_(606)S_(85),相对分子质量为38808.87,理论等电点为5.25;无信号肽和跨膜区,二级结构包含α-螺旋(81.01%)、无规则卷曲(13.19%)、β-折叠(3.16%)和延伸链(1.90%);含有9个线性B淋巴细胞抗原表位和5个T淋巴细胞抗原表位,以及10个磷酸化位点和10个互作蛋白,Bfr和Bfrd蛋白表面匹配良好、结合稳定。试验成功克隆出Bfr基因,其大小约为476 bp,获得大小约为35 ku的高纯度重组蛋白Bfr。说明Bfr蛋白具有良好的反应原性,有可能成为鼠伤寒沙门氏菌的疫苗开发和疾病诊断的候选蛋白。In order to obtain high-purity Salmonella typhimurium bacterial ferritin(Bfr),in the experiment,the structure,post-translational modifications and antigenic epitopes of Bfr were predicted and analyzed using bioinformatics software;the Bfr gene of Salmonella typhimurium was amplified by PCR,ligated with pET-32a(+)vector and transformed into Escherichia coli BL21(DE3)strain,and the expressed and purified target protein was detected by SDS-PAGE analysis and Western-blot.The results showed that Bfr consisted of 476 amino acids with molecular formula C_(1440)H_(2406)N_(476)O_(606)S_(85),relative molecular weight 38808.87 and theoretical isoelectric point 5.25;without signal peptide and transmembrane region,the secondary structure containedα-helix(81.01%),random coil(13.19%),β-fold(3.16%)and extended chain(1.90%).The 9 linear B lymphocyte antigenic epitopes and 5 T lymphocyte antigen epitopes were contained,as well as 10 phosphorylation sites and 10 reciprocal proteins;Bfr and Bfrd proteins were well-matched on the surface and stable in binding.The test successfully cloned the Bfr gene,which was about 476 bp in size,and obtained the high-purity recombinant protein Bfr with a size of about 35 ku.The results indicated that Bfr protein had good reactogenicity and might be a target candidate protein for vaccine development and disease diagnosis of Salmonella typhimurium.

关 键 词：鼠伤寒沙门氏菌 细菌铁蛋白(Bfr) 生物信息学 原核表达 SDS-PAGE 

分 类 号：S852.23[农业科学—基础兽医学]