禽源肠炎沙门菌aroA基因缺失株的构建及其生物学特性研究

试验旨在研究aroA基因对肠炎沙门菌生物学特性及毒力的影响。利用λ-Red同源重组技术，将从田间分离的禽源肠炎沙门菌的aroA基因进行敲除，经连续传代检测缺失株遗传稳定性，并通过细菌生长曲线、对不同生化反应管的利用情况、生物膜形成能力、对环境应激的抵抗能力及对1日龄雏鸡的毒力比较野生株和基因缺失株之间的差异。结果显示，试验成功构建缺失株，连续传30代未发现目的基因回复突变；在相同培养条件下，缺失株与野生株在相同时间进入对数生长期和稳定期，但缺失株生长速率低于亲本株；缺失株较野生株生化特性未发生改变；缺失株的生物膜形成能力及对酸、碱、高渗和热应激的抵抗能力均显著低于野生株（P<0.05）；1日龄雏鸡分别滴口接种缺失株和野生株，观察14 d，野生株组鸡全部死亡，而缺失株组无死亡。综上所述，本试验成功构建并获得了具有良好遗传稳定性的肠炎沙门菌aroA基因缺失株，缺失株生化特性和体外生长趋势未发生明显改变，但生物膜形成能力、对环境应激抵抗能力及毒力均明显下降。本研究为进一步研究肠炎沙门菌aroA基因缺失株的免疫原性奠定了基础。

Construction and Biological Characteristics of aro A Gene Deletion Strain of Salmonella Enteritis from Poultry

The puopose of the study was to analyze the effect of aroA gene on the biological characteristics and virulence of Salmonella Enteritidis.The aroA gene of Salmonella Enteritis isolated from poultry in the field was knocked out using λ-Red homologous recombination.The genetic stability of the gene deletion strain was tested by continuous passages.The differences between the wild strain and the gene deletion strain were compared by bacterial growth curve,the utilization of different biochemical reaction tubes,the ability of biofilm formation,the resistance to environmental stress,virulence to 1-day-old chicks.The results showed that the gene deletion strain was successfully constructed,and no revert mutation was found in the target gene after 30 successive generations.The deletion strains entered the logarithmic growth phase and stable phase at the same time compared with the wild strains under the same culture conditions,but the growth rate of the deletion strains was lower than that of the parent strains.The biochemical characteristics of the deletion strains were not changed compared with the wild strains.The ability of biofilm formation and resistance to acid,alkali,high osmolar and heat stress of the deletion strain were significantly lower than those of the wild strain (P<0.05).1-day-old chicks were inoculated through the mouth with the deletion strain and the wild strain respectively.All the chickens in the wild strain group were died after 14 days of observation,while none in the deletion strain group.In conclusion,the aroA gene deletion strain of Salmonella Enteritis with good genetic stability was successfully constructed and obtained in this experiment.The biochemical characteristics and in vitro growth trend of the deletion strain were not significantly changed,but the biofilm formation ability,resistance to environmental stress and virulence were significantly decreased.This study laid a foundation for the further study of the characteristics and immunogenicity of aroA gene in Salmonella Enteritis.