一株肠炎沙门氏菌鞭毛基因FliC的克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2018.12.009

摘要/Abstract

摘要：

本研究旨在对肠炎沙门氏菌（Salmonella Enteritidis）鞭毛蛋白FliC基因进行克隆，并对所获序列进行生物信息学分析。提取该菌基因组DNA作为模板，参考GenBank中肠炎沙门氏菌FliC基因序列设计1对引物，利用PCR克隆获得FliC基因，将其插入到克隆载体中进行测序，应用生物信息学软件分析该基因的核苷酸和氨基酸序列，利用BLAST进行同源性比对，并构建系统进化树，同时对该基因编码蛋白的理化性质、亲水性、信号肽、跨膜结构域、糖基化位点和B细胞抗原表位、二级结构、三级结构等进行分析。结果显示，试验成功克隆了1 518 bp的目的基因，编码505个氨基酸。同源性比对发现，FliC基因相对保守，与大肠杆菌属有较高的同源性。该蛋白的化学分子式为C₂₂₅₄H₃₇₀₁N₆₅₇O₈₀₃S₄，理论分子质量为52.981 ku，理论等电点为4.91；不稳定指数为16.86，是稳定存在的亲水蛋白质。结构分析结果显示，该蛋白没有信号肽或跨膜结构域，但具有8个N-糖基化位点、13个O-糖基化位点和60个磷酸化位点，同时还含有26个B细胞线性结合位点和5个T细胞结合位点。二级结构分析显示，α-螺旋、β-转角、延伸链和无规卷曲分别占43.76%、3.76%、20.99%和31.49%。本试验成功克隆了肠炎沙门氏菌鞭毛基因FliC，并对其序列结构进行了分析，为进一步研究鞭毛在肠炎沙门致病过程中的作用及基因工程疫苗的研制提供理论依据。

关键词: 肠炎沙门氏菌; 鞭毛; FliC基因; 生物信息学分析

Abstract:

This study was aimed to clone the flagellin FliC gene of Salmonella Enteritidis and analyze its genetic structure with bioinformatics.The genomic DNA of bacterium was extracted as a template,according to the published sequence of FliC gene in GenBank,and one pair of specific PCR primers was designed.FliC gene was cloned by PCR and linked into the cloning vector to be sequenced.The nucleotide sequence,amino acid sequence,homology and phylogenetic tree were analyzed by the bioinformatics softwares.The physicochemical properties,hydrophilicity,signal peptides,transmembrane domains,glycosylation sites,B cell epitopes,secondary structure and tertiary structure of the encoding protein were predicted by DNAStar software and online servers.The results showed that the gene of 1 518 bp was successfully cloned and encoded 505 amino acids.Homology analysis showed that FliC gene was relatively conserved,and this species had high homology with E.coli genus.The chemical formula of this protein was C₂₂₅₄H₃₇₀₁N₆₅₇O₈₀₃S₄,and the theoretical molecular mass was 52.981 ku.The theoretical isoelectric point was 4.91,and the instability index was 16.86,which was a stable hydrophilic protein.Structural analysis result showed that neither a signal peptide nor a transmembrane domain was found,however,a total of 8 N-linked glycosylation sites,13 O-linked glycosylation sites,60 phosphorylation sites,26 linear B cell piptopes and 5 T cell binding sites were predicted.Secondary structure analysis showed that α-helix,β-turn,extended chain and random coil accounted for 43.76%,3.76%,20.99% and 31.49%,respectively.In this study,FliC gene was cloned and analyzed successfully.The results laid a theoretical foundation for the further study of the role of flagella in the pathogenesis and the development of genetic engineering vaccines.

Key words: Salmonella Enteritidis; flagella; FliC gene; bioinformatics analysis

中图分类号:

S855.1⁺2

程相朝, 田文静, 张梦珂, 毛福超, 廖成水. 一株肠炎沙门氏菌鞭毛基因FliC的克隆及生物信息学分析[J]. 《中国畜牧兽医》, 2018, 45(12): 3379-3386.

CHENG Xiangchao, TIAN Wenjing, ZHANG Mengke, MAO Fuchao, LIAO Chengshui. Cloning and Bioinformatics Analysis of a Flagellar FliC Gene in Salmonella Enteritidis[J]. , 2018, 45(12): 3379-3386.

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