摘要
目的本研究旨在探究sifA基因对肠炎沙门菌胞内寄生和增殖的影响,以探索肠炎沙门菌的致病机理。方法根据同源重组原理,无痕构建肠炎沙门菌C50041的sifA基因缺失株,通过蓝白斑、抗生素耐药性、PCR和基因测序方法对基因缺失株进行鉴定,并从生物学特性、胞内沙门菌增殖、sifA基因在胞内外菌中表达等方面,分析sifA基因对肠炎沙门菌的影响。结果成功构建肠炎沙门菌C50041ΔsifA,蓝白斑、抗生素耐药性、PCR和基因测序方法证明该菌株是正确的。其生长速度和生化特性没有发生改变。ΔsifA株感染巨噬细胞后,其胞内沙门菌量显著少于其野生株。sifA基因在spiC基因缺失株中表达量增加。结论肠炎沙门菌C50041ΔsifA被成功构建,其在巨噬细胞内的增殖量显著降低,sifA基因表达可能受spiC基因调控,本研究为深入探究肠炎沙门菌在细胞内增殖及sifA基因的功能奠定基础。
Salmonella spp.is one of facultative intracellular pathogens.SCV provides a safe environment for intracellular Salmonella,enabling it to survive in macrophages without being cleared.The sifA gene involves information of SCV and promotes the survival and replication of intracellular Salmonella.We aimed to study the effect of sifA gene on intracellular proliferation of Salmonella Enteritidis and further to explore its pathogenic mechanism.The C50041ΔsifA were constructed by homologous recombination,it was verified by blue-white clones,antibiotic resistances and PCR.And assays of growth characteristics,biochemical characteristics,intracellular proliferation of C50041ΔsifA and sifA gene expression were performed.C50041ΔsifA was displayed that there was no difference in biochemical characteristics and growth speed,but its ability of survival and proliferation of C50041ΔsifA decreased in macrophages RAW264.7 compared to that of parental C50041.Whether inside or outside of macrophages RAW264.7,mRNA of sifA gene increased in the spiC gene mutant by qPCR.It was concluded that Salmonella Enteritidis C50041ΔsifA was successfully constructed,its proliferation could significantly decrease in macrophages and sifA gene expression might be regulated by spiC gene,which would lay an important academic foundation for in-depth research on proliferation in macrophages and the function of the sifA gene.
作者
刘桂凤
杨家兴
丁炜
王耀楠
蔡媛
张健
王洋
耿士忠
潘志明
焦新安
LIU Gui-feng;YANG Jia-xing;DING Wei;WANG Yao-nan;CAI Yuan;ZHANG Jian;WANG Yang;GENG Shi-zhong;PAN Zhi-ming;JIAO Xin-an(Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality,Ministry of Agriculture of China/Key Laboratory of Zoonoses of Jiangsu Province/Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2020年第3期178-186,共9页
Chinese Journal of Zoonoses
基金
国家重点研发项目(No.2016YFD0501607)
江苏省重点研发项目(No.BE2018334)
江苏省自然科学基金(No.BK20151306)
江苏省重点实验室开放课题项目(No.0273896034819)资助。
关键词
肠炎沙门菌
同源重组
sifA基因
胞内增殖
Salmonella Enteritidis
homologous recombination
sifA gene deletion
intracellular proliferation
