摘要
目的为深入研究鼠伤寒沙门菌毒力基因spvB的功能,制备鼠伤寒沙门菌spvB基因完全缺陷变异株,观察spvB基因缺陷株在体外酸性环境中的生存能力。方法根据鼠伤寒沙门菌spvB基因序列,设计PCR特异性引物,制备spvB基因缺陷性同源性核苷酸片段,导入自杀质粒pCVD442后再导入鼠伤寒沙门菌野生株,进行同源重组,用PCR方法观察重组现象,将完全重组的菌株作为spvB基因的缺陷变异株,并通过核苷酸序列分析加以确定。绘制生长曲线,对比spvB基因缺陷变异株与野生株在酸性条件下的生存情况。结果 PCR及序列分析证实,缺陷变异株的spvB基因缺失1 749bp。酸性条件下培养1h及2h后,野生株的活菌数高于spvB基因缺陷株,差异有统计学意义(P<0.05);且野生株在酸性环境中的生存率明显高于缺陷株,野生株在1h及2h后的生存率分别为85.6%、74.9%,缺失株分别为68.0%、42.3%。结论成功构建鼠伤寒沙门菌spvB基因缺陷变异株,并发现在酸性条件下其生存能力明显降低,为进一步研究其在鼠伤寒沙门菌中的功能奠定了基础。
Objective To investigate the function of spvB gene,a toxicity gene of Salmonella enterica serovar Typhi,by constructing the spvB gene-deleted mutant and examinimg its survival ability in acid condition.Methods According to the sequences of spvB gene of Salmonella enterica serovar Typhi,specific primers were designed for PCR.The homologous DNA fragments with spvB gene deleted were constructed,which was cloned into the suicide plasmid pCVD442 and then transferred into the target cells of Salmonella enterica serovar Typhi.The recombination was visualized by PCR,and the complete recombinant strain was selected as the spvB gene-deleted mutant strain and confirmed by the corresponding sequencing analysis.Under the acid condition,the survival ability of the spvB mutant and parent was compared by using the growth curve.Results A deletion of 1 748 bp of the spvB gene was confirmed by PCR and sequencing analysis in spvB gene-deleted mutants.The number of live wild-type strains were significantly greater than that of spvB gene-deleted mutants under the acid condition for 1and 2h,and the difference was statistically significant（P〈0.05）.The survival rate of the wild-type strains was 85.6%and 74.9%at 1and 2h,significantly higher than that of the spvB gene-deleted mutants,which was 68.0%and 42.3%.Conclusion The spvB gene-deleted mutant of Salmonella enterica serovar Typhi was successfully generated and its survival ability was significantly compromised under the acid condition,which lays a foundation for studying the function of the spvB gene in Salmonella enterica serovar Typhi.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2014年第4期399-404,共6页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
江西省自然科学基金资助项目(No.20132BAB205036)
关键词
鼠伤寒沙门菌
spvB
基因缺陷变异
同源重组
生长曲线
Salmonella enterica serovar Typhi
spvB
gene-deleted mutation
homologous recombination
growth curve
