A gene knock-in method used to purify plasmid pSPI12 from Salmonella enterica serovar Pullorum and characterization of IpaJ

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• 作者：Qiuchun Li ; Yachen Hu ; Yaohui Xu ; Jing Chen ; Lijun Fang ; Zhicheng Liu ; Xinan Jiao ; ^{jiao@yzu.edu.cn" class="auth_mail}
• 关键词：PD ; Pullorum disease ; IPTG ; Isopropyl 尾-d-1-thiogalactopyranoside ; ECL ; Enhanced chemiluminescence ; DMEM ; Dulbecco's modified Eagle medium ; MOI ; Multiplicity of infection
• 刊名：Journal of Microbiological Methods
• 出版年：March, 2014
• 年：2014
• 卷：98
• 期：Complete
• 页码：128-133
• 全文大小：547 K

文摘

A small plasmid with 4080 bp long, designated pSPI12, was purified from Salmonella enterica serovar Pullorum using a gene knock-in method by inserting a kanamycin resistance cassette in the plasmid. The G + C content of the plasmid was 51.8%, which is in the range of Salmonella genomic DNA. A sequence analysis revealed that pSPI12 had 99.1% homology to pSFD10, which was first reported in the vaccine strain S. enterica serovar Chloreaesuis C500, but not prevalent among other strains of S. Chloreaesuis. The plasmid has seven open reading frames (ORFs), with one ORF containing a putative virulence-related protein, which had 49% homology with invasion plasmid antigen J protein (IpaJ) secreted by type III secretion system of Shigella flexneri. The putative IpaJ protein was expressed and purified as a His-tagged fusion protein reacted with convalescent sera against S. Pullorum, confirming its identification as an immunogen of the pathogen. In addition, the gene was upregulated for 1 h post-infection of HD-11 cells with the pathogen by a quantitative real-time reverse transcription PCR assay. The results suggest that IpaJ may be a virulent protein involved in the early stage of infection by S. Pullorum.