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[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] The purpose of this study was to knock out the virulence gene spvC of Salmonella typhi with λ Red recombination system. [Method] Firstly, plasmid p KD4 was used as a template to amplify the linear DNA fragment containing the spvC homology arms on both sides and the kanamycin resistance gene in the middle. Then this linear fragment was electroporated into recombinant competent Competent salmonella strains. After recombination, kanamycin plates were screened for positive transformants. Finally, the kanamycin resistance gene between FRT sites was eliminated using plasmid pCP20, which expresses the FLP recombinase, and was identified by PCR. Western Blot detection of wild-type Salmonella and spvC knock-out infected Hela cells ERK phosphorylation levels. [Results] The virulence gene of Salmonella spvC knockout was successfully constructed and the level of ERK phosphorylation in He La cells infected by spvC knockout strain was increased. [Conclusion] The spvC knockout strain of Salmonella typhi plasmid virulence gene was successfully constructed and the function of spvC gene was verified.