摘要
研究鼠伤寒沙门氏菌(S.typhimurium)sopF基因对细菌生理特性的影响及在感染巨噬细胞时的作用。应用CRISPR/Cas9系统对S.typhimurium sopF基因进行敲除,PCR扩增得到sg20片段和sopF上下游同源臂片段,利用重叠PCR连接3个DNA片段,并与pTargetF质粒连接后,获得敲除质粒pTargetF-sopF;将pCas与重组质粒PtargetF-sopF电转入S.typhimurium以敲除sopF基因。利用生长曲线测定确定sopF基因对细菌生长的影响,Western blot和免疫荧光试验研究该基因对细菌引起的宿主自噬的影响,CCK8检测该基因对细菌引起的细胞毒性影响。结果显示,S.typhimuriumΔsopF突变体构建成功,sopF基因的缺失并不影响S.typhimurium的生长,但在感染宿主细胞时,会诱导更强的细胞自噬反应,同时导致更弱的细胞毒性,说明sopF基因对S.typhimurium的毒力有影响,为后续用CRISPR/Cas9系统构建S.typhimurium突变体及研究sopF基因功能提供参考。
To investigate the role of Salmonella enterica serovar typhimurium(S.typhimurium)sopFge-neinits physiological characteristics and infection in macrophages,the CRISPR/Cas9 system was used to generate the OsopF mutant.Fragments of sg20,upstream(Up)and downstream(Down)of sopF were ob-tained by PCR.Three fragments were overlapped by PCR to generate sg20-Up-Down and then the product was ligated into the pTargetF plasmid to construct the knockout plasmid pTargetF-sopF.The pCas and the PtargetF-sopF were electroporated into S.typhimurium to knock out the sopF gene.The effect of the gene on bacterial growth was determined by the growth curve assay,W estern blot and immunofluorescence as-says were used to determine the role of sopFon S.typh imurium induced-autophagy,CCK8 assay was used to detect the character of sopFin the bacterial-induced cytotoxicity.The results showed that the OsopF mu-tant was successfully constructed,and the deletion of the sopF did not change the growth rate of S.typhi-murium,but could induce stronger autophagy and weaker cytotoxicity during infection,w hich indicates that the sopF gene could affect the virulence of S.ty phimurium.Together,this research provides a reference for the S.typhimurium mutant construction by CRISPR/Cas9,and also provides a promising tool in investiga-ting the role of sopF.
作者
李梦园
王逍
杨亚东
汤晶晶
谢臻
徐磊
沈锡辉
王瑶
LI Meng-yuan;WANG Xiao;YANG Ya-dong;TANG Jing-jing;XIE Zhen;XU Lei;SHEN Xi-hui;WANG Yao(Collegeof Life Science,Northwest A&F University,Yangling,Shaanxi,712100,China)
出处
《动物医学进展》
北大核心
2023年第3期1-7,共7页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31725003,31800113)
中国博士后科学基金特别资助项目(2020T130543)
中国博士后科学基金面上资助项目(2018M631201)。
