鼠伤寒沙门菌SL1344株△crp△sipB缺失株生物学特性的检测

Biological characteristics of the △crp△sipB mutant strain of Salmonella typhimurium SL1344

目的进一步深入研究△crp△sipB双基因缺失株的减毒机制。方法以鼠伤寒沙门菌亲本菌株SL1344和△crp△sipB双基因缺失株为研究对象，利用细菌在半固体培养基上形成的运动圈进行流动性的检测，通过比色法测定出乳酸、丙酮酸的含量以及上皮细胞增殖快慢。结果在半固体培养基上双缺失株形成的运动圈直径与亲本菌株相比发生了显著的降低，表明缺失株的运动能力弱于亲本株；对培养不同时间点的缺失株和亲本株菌体的丙酮酸、乳酸测定结果显示：缺失株菌体内的丙酮酸含量在培养后6、9和12h均高于亲本株组（P〈0．05），乳酸含量在9h以后也高于亲本株组（P〈0．05），结果表明缺失株的糖酵解能力高于亲本株组；对HeLa细胞的增殖和毒性试验结果表明，在感染细胞后各个时间点△crp△sipB基因缺失株组的A530值均高于亲本菌株组（P〈0．05），说明双基因缺失后对细胞增殖和细胞毒性的影响均弱于亲本菌株；△crp△sipB基因缺失后对上皮细胞的黏附和侵入能力与亲本菌株相比发生显著降低（P〈0．05）。结论上述结果为深入研究以鼠伤寒沙门菌为载体的口服多价疫苗奠定基础。

Objective To investigate the mechanism of attenuated toxicity in the △crp△sipB mutant strain. Methods The mutant strain of Salmonella typhimurium （S. typhimurium） SL1344 with crp and sipB genes deletion （△crp△sipB） and its parent SL1344 strain were used in this study. The two types of strains were comparatively analyzed on several parameters including motility, contents of pyruvic acid and lactic acid and adhesion ability. Results Compared with the parent SL1344 strain, the △crp△sipB mutant strain showed significantly decreased motility. The levels of pyruvic acid in the △crp△sipB mutant strain were higher than those in the parent strain after culturing for 6 h, 9 h and 12 h （P〈0.05）. The levels of lactic acid in the △crp△sipB mutant strain were higher than those in the parent strain after culturing for 9 h and 12 h （P〈0.05）. It indicated that the bacterial glycolysis was enhanced in the △crp△sipB mutant strain. Compared with the parent SL1344 strain, the △crp△sipB mutant strain showed weakened cytotoxicity and decreased inhibitory effects on the proliferation of Hela cells. The adhesion and invasion capabilities of the S. typhimurium SL1344 strain were weakened after the deletion of crp and sipB genes. Conclusion This study has paved the way for further investigation on safe and effective attenuated S. typhirnurium vaccine.