肠炎沙门氏菌tu-fA基因缺失株的构建及其生物学特性研究

Construction and biological identification of a tu-fA gene deletion mutant from Salmonella enterica

为构建肠炎沙门氏菌(S.enterica)tu-fA基因缺失株及鉴定其生物学特性,本实验利用Red重组系统,以pKD3中的FRT-氯霉素抗性(cat^r)基因-FRT序列为模板,利用含有tu-fA基因两端序列的引物,经PCR扩增同源重组DNA片段(tu-fA5'-FRT-cat^r-FRT-tu-fA3'),将该DNA片段电转化于含有质粒pKD46的S.enterica SM6株中进行同源重组,通过氯霉素抗性筛选tu-fA基因缺失的SM6株(SM6△tu-f A::cat),再通过导入质粒pCP20于SM6△tu-fA::cat中敲除cat r基因,从而构建了tu-fA基因缺失株SM6△tu-fA。生物学特性鉴定结果表明,基因缺失株具有良好的遗传稳定性;与亲本株相比,细菌形态未发生明显改变,但基因缺失株体外生长速率略低,利用碳源的能力有所增强;缺失株侵袭Caco-2细胞的能力与亲本株相比有所降低,表明tu-f A基因编码的延伸因子(EF-Tu)在调控细菌侵袭细胞的过程中起着一定的作用。本研究通过构建tu-fA基因缺失的S.enterica株,为进一步研究tu-fA基因编码的EF-Tu的功能奠定基础。

To construct the tu-fA gene deleted Salmonella enterica and identify the biological characteristics of the mutant, the chloramphenicol resistance （cat＇） gene flanked by homologous sequence of the S.entericatu fA gene was amplified by PCR from plasmid pKD3 and electro-transformed into S.enterica SM6 strain containing pKD46 to replace tu-fA gene by the catr gene to construct the mutant of SM6Atu-fA：：cat. In addition, the catr gene was eliminated from the SM6Atu-fA：：cat to generate the SM6A tu-fA by transforming the suicide plasmid ofpCP20 and incubating at 42 ℃. The identification results showed that the SM6Atu-fA strain had high genetic stability and morphology of SM6Atu-fA strain had no significant difference with the parental strain, whereas, comparing with the parental strain, the SM6Atu-fA strain was slightly slow in growth rate, increased the ability of consuming carbon source, and decreased the invasive ability to the Caco-2 cells, which indicated that the EF-Tu （encoded by tu-f_A gene） plays a role in the process of regulating bacteria invade cells. These results provided a basis for further study on the function of the EF-Tu.