目的:探讨肠炎沙门菌n spvD基因对人克隆结肠腺癌Caco-2细胞侵袭力及胞内增殖力的影响。n 方法:利用自杀质粒介导的同源重组技术构建n spvD基因缺失株，PCR扩增并克隆n spvD基因于pBAD33表达载体获得回补质粒，导入相应缺失株得到回补株，并将pBAD33导入野生株及缺失株作为空载对照。荧光定量反转录聚合酶链式反应检测n spvD mRNA表达水平。以Caco-2细胞作为体外模拟人肠上皮细胞模型，分别将野生株、缺失株及回补株与其共培养，探究n spvD基因对Caco-2细胞的毒力。使用LSD-n t检验进行3组间n spvD mRNA表达水平比较及胞内活菌数比较，使用χ2检验进行3组间侵袭率的比较。n 结果:以野生株n spvD mRNA表达量作为单位“1”，缺失株为“0.00”、回补株为“2.60”（LSD-n t野生株，缺失株=1.11，n P=0.31；LSD-n t野生株，回补株=-1.77，n P=0.13；LSD-n t缺失株，回补株=-2.88，n P=0.03），该结果证实了缺失株及回补株的成功构建。上述3组肠炎沙门菌对Caco-2细胞的侵袭实验结果显示，野生株侵袭率为0.23%，缺失株侵袭率为0.16%，回补株侵袭率为0.16%，差异无统计学意义（χn 2=1.13，n P=0.570）。通过比较细菌干预Caco-2细胞后不同时间点胞内活菌数，发现在干预16 h时，野生株（6.50×10n 6 CFU/ml）、回补株（7.25×10n 6 CFU/ml）胞内活菌数明显增多，均高于缺失株（1.90×10n 6 CFU/ml）（LSD-n t野生株，缺失株=7.95，n P=0.00；LSD-n t野生株，回补株=-1.27，n P=0.25；LSD-n t缺失株，回补株=-9.22，n P=0.00）。n 结论:尚不能认为n spvD基因影响肠炎沙门菌对Caco-2细胞的侵袭力，但该基因可促进肠炎沙门菌在Caco-2细胞内增殖。n “,”Objective:To analyze the effects of n spvD gene on invasion and intracellular proliferation of Caco-2 cells and in order to provide insight into the function of that gene and the underlying mechanism of n Salmonella caused infection.n Methods:Functional verification of n spvD gene deletion mutant and compensation strain. The deletion mutant strain was constructed through a suicide plasmid-mediated homologous recombination. The compensation plasmid constructed by cloning the coding sequence of n spvD by PCR into plasmid pBAD33 was mobilized into the deletion mutant by conjugation and the pBAD33 was introduced into wild strains and deleted mutant strains as control. The relative expression of n spvD mRNA was detected by quantitative reverse transcription PCR. In order to analyze the virulence of n spvD against Caco-2 cells, Caco-2 cells was cocultured with wild type n Salmonella enteritidis carrying n spvD gene, the deletion mutant strain and compensation strain respectively. The expression level of n spvD mRNA and the the number of n Salmonella enteritidis after Caco-2 cells intervention were compared between the three groups by LSD-n t test, and the invasion rate was compared by χn 2 test.n Results:The expression level of n spvD mRNA in wild type n Salmonella enteritidis was set as unit “1”, the deletion mutant strain was “0.00”, and the compensation strain was “2.60” (LSD-n twild, deleted=1.11, n P=0.31; LSD-n twild, compensation=-1.77, n P=0.13; LSD-n tdeleted, compensation=-2.88, n P=0.03), which confirmed the successful construction of the deletion mutant strain and the compensation strain. The invasion experiment results of the above three n Salmonella enteritidis strains on Caco-2 cells showed that the invasion rate of wild strain was 0.23%, the invasion rate of deleted mutant strain was 0.16%, and the invasion rate of compensation strain was 0.16%, with no statistical significance (χn 2=1.13, n P=0.570). By comparing the number of n Salmonella enteritidis at different time points after Caco-2 cells intervention, it was discovered that the number of n Salmonella enteritidis in wild strains (6.50×10n 6 CFU/ml) and compensation strains (7.25×10n 6 CFU/ml) was significantly increased than that in deletion mutant strain (1.90×10n 6 CFU/ml) after 16 h coculture (LSD-n twild, deleted=7.95, n P=0.00; LSD-n twild, compensation=-1.27, n P=0.25; LSD-n tdeleted, compensation=-9.22, n P=0.00).n Conclusion:It is not considered that n spvD gene can affect the invasion of n Salmonella enteritidis on Caco-2 cells, but the gene can promote the reproduction of n Salmonella enteritidis in Caco-2 cells.n