【摘要】 [目的]利用λRed重组系统敲除沙门菌质粒毒力基因spvC。[方法]首先以质粒p KD4为模板,扩增得到两侧含spvC同源臂、中间为卡那霉素抗性基因的线性DNA片段。再将此线性片段电转入具重组功能的感受态沙门菌菌株,发生重组后,卡那霉素平板筛选阳性转化子。最后利用表达FLP重组酶的质粒p CP20,将FRT位点之间的卡那霉素抗性基因消除,用PCR鉴定。Western Blot检测野生沙门菌和spvC敲除株感染的He La细胞ERK磷酸化水平。[结果]沙门菌质粒毒力基因spvC敲除株构建成功,spvC敲除株感染的He La细胞内ERK磷酸化水平升高。[结论]成功构建沙门菌质粒毒力基因spvC敲除株,验证了spvC基因的功能。更多还原

【Abstract】 [Objective]To construct Salmonella mutant spvC gene knockout by λRed recombination system. [Methods]First,the linear DNA fragment containing spvC homology arms and kanamycin resistance gene was amplified,using the plasmid p KD4 as template. Next,this linear DNA fragment was transformed into competent cells by electroporation,and the positive transformants were selected by LB plate containing kanamycin. Finally,eliminated the kanamycin resistance gene which located between the FRT sites,using the plasmid p CP20 expressing Flp recombinase,and finally got spvC gene knockout strains by PCR analysis. The phosphorylation levels of ERK in He La cells that infected by wild type Salmonella and ΔspvC mutants were detected by Western Blotting analysis. [Results]Salmonella mutant with spvC gene knockout was successfully constructed,which could enhance the phosphorylation level of ERK. [Conclusion]The Salmonella mutant with spvC gene knockout was constructed successfully,and the function of spvC was verified.更多还原