λRed重组系统用于沙门菌质粒毒力基因spvC敲除株的构建  

Construction of Salmonella mutant with spvC gene knockout by λRed recombination system

作　　者：高嵩[1] 燕婧 储元元[1] 李嫄渊[1] 吴淑燕[1] 

机构地区：[1]苏州大学基础医学与生物科学学院病原生物学系,江苏苏州215123

出　　处：《生物技术》2016年第4期367-371,共5页Biotechnology

基　　金：江苏省高校研究生科研创新计划("沙门菌质粒毒力基因spvC对细胞自噬及体内感染过程的影响";No.KYLX_1226)

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。[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 pKD4 as template. Next, this linear DNA fragment was transformed into competent cells by electroporation, and the positive transforma- nts were selected by LB plate containing kanamycin. Finally, eliminated the kanamycin resistance gene which located between the FRT sites, using the plasmid pCP20 expressing Flp recombinase, and finally got spvC gene knockout strains by PCR analy- sis. The phosphorylation levels of ERK in HeLa cells that infected by wild type Salmonella and AspvC mutants were detected by Western Blotting analysis. [ Results] Salmonella mutant with spvC gene knockout was successfully constructed,which could en- hance the phosphorylation level of ERK. [ Conclusion ] The Salmonella mutant with spvC gene knockout was constructed successfully, and the function of spvC was verified.

关 键 词：λRed重组系统 沙门菌 SPVC 基因敲除 质粒 

分 类 号：R378.22[医药卫生—病原生物学]