【摘要】 为探究鼠伤寒沙门菌（Salmonella Typhimurium）VI型分泌系统（T6SS）分泌的效应蛋白Rhs对该菌生物学特性的影响，本研究利用Red同源重组技术构建了鼠伤寒沙门菌rhs1和rhs2基因缺失株CVCC541Δrhs1和CVCC541Δrhs2，同时构建了基因回补菌株C-Δrhs1和C-Δrhs2，并经PCR和测序鉴定正确后，测定了鼠伤寒沙门菌亲本株和缺失株的遗传稳定性、生长曲线，结果显示，缺失株经过多次传代后仍然具有稳定的遗传性；Rhs效应因子缺失对细菌的生长未造成明显影响。进一步利用各菌株感染Hela细胞，对菌株黏附力和侵袭力进行测定，结果显示，与亲本株相比，缺失株CVCC541Δrhs1和CVCC541Δrhs2对Hela细胞的黏附力无明显影响，但侵袭力分别提高了4.9倍和3.4倍。利用各菌株感染RAW264.7巨噬细胞，检测各株菌在RAW264.7细胞内的存活力，结果显示缺失株CVCC541Δrhs1和CVCC541Δrhs2在RAW264.7细胞内存活力分别提高了7.8倍和5.9倍。将各菌经株腹腔注射感染小鼠，检测其对小鼠的半数致死量(LD₅₀)，结果显示，亲本株的LD₅₀为2.53×10⁶cfu/mL，缺失株CVCC541Δrhs1、CVCC541Δrhs2的LD₅₀分别为1.31×10⁷cfu/mL和5.40×10⁶cfu/mL,LD₅₀分别下降了5.2倍和2.1倍。本研究首次证实效应蛋白Rhs会影响鼠伤寒沙门菌的侵袭力和细胞内的生存能力，同时也会降低细菌的毒力，在鼠伤寒沙门菌感染过程中发挥了作用，该结果为阐释鼠伤寒沙门菌的致病机制提供了参考。更多还原

【Abstract】 To investigate the influence of effector protein Rhs secreted by Type VI secretion system （T6SS） of Salmonella Typhimurium on its biological characteristics,Salmonella Typhimurium rhs1 and rhs2 gene deletion strains CVCC541Δrhs1 and CVCC541Δrhs2 were constructed by Red homologous recombination technique,and the complement strains C-Δrhs1 and C-Δrhs2were constructed and identified by PCR and sequencing.The genetic stability and growth curves of the parent and mutant strains were determined.The results showed that the mutant still had stable heritability after continuous passages.The absence of Rhs effector had no significant effect on the growth of the bacteria.Hela cells were further infected with teach strain,and the adhesion and invasion of the strains were determined.The results showed that,compared with their parents,the deletion strains CVCC541Δrhs1 and CVCC541Δrhs2 had no significant effect on the adhesion of Hela cells,but the invasiveness was increased by4.9 and 3.4 times,respectively.The survival ability of each strain in RAW264.7 cells and the 50%lethal dose (LD₅₀) in mice were investigated.It showed that the CVCC541Δrhs1 and CVCC541Δrhs2 survived in RAW264.7 cells increased by 7.8 and 5.9 times,respectively,and the LD₅₀of the parent strain and the two mutants was 2.53×10⁶cfu/mL,1.31×10⁷cfu/mL and 5.40×10⁶cfu/mL,respectively.The virulence of each mutant was decreased by 5.2 and 2.1 times,respectively.This study confirmed for the first time that the effector protein Rhs can affect the invasiveness and intracellular viability of Salmonella Typhimurium,and also reduce the virulence of the bacteria,which plays a role in the infection process of Salmonella Typhimurium,providing a reference for the interpretation of the pathogenic mechanism of Salmonella Typhimurium.更多还原