表皮葡萄球菌serp2169基因敲除突变株的构建及其生长变化

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摘要目的探讨表皮葡萄球菌serp2169基因敲除对其生物学功能的影响。方法构建含有壮观霉素抗性基因(spc)的同源重组质粒pMAD- serp2169,电转入表皮葡萄球菌1457株,在42 ℃条件下振摇培养,利用蓝白斑和抗生素抗性筛选表皮葡萄球菌serp2169基因敲除突变株(SE1457-Δserp2169),通过检测OD₆₀₀值观察其生长曲线的变化,同时检测其培养液的pH值,微量板半定量方法检测serp2169敲除突变对细菌生物膜形成的影响。结果成功构建同源重组质粒pMAD- serp2169。经PCR扩增和测序验证获得了表皮葡萄球菌serp2169基因敲除突变株。serp2169突变株与野生株的生长曲线在平台期出现了差异,野生株有一个二次生长的过程,而突变株则没有。两株细菌形成生物膜的能力无显著差异。结论表皮葡萄球菌serp2169基因可调控平台期细菌的生长,但对生物膜的形成无影响。

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	李朝品

关键词 ：表皮葡萄球菌, 同源重组, serp2169基因

Abstract：To investigate the biological function of serp2169 gene in S. epidermidis, a plasmid pMAD- serp2169 encompassing the upstream and downstream homologous region of serp2169 and spectinomycin resistance gene (spc) was constructed, and then transformed into S. epidermidis 1457 by electroporation, continuously subcultured at 42 ℃ with shaking. The serp2169 knockout mutant (SE1457-Δserp2169) was selected by blue-white colony screening and antibiotic resistance. Bacterial growth curve was determined by measuring the value of OD₆₀₀, and the pH value of culture medium was also examined. Effect of the serp2169 mutant on biofilm formation was detected by a semi-quantitative microtiter plate assay. The plasmid pMAD- serp2169 was constructed and serp2169 was deleted from the genome of S. epidermidis 1457. The knockout mutant was verified by PCR amplification and sequencing. The growth curve of the wild type strain indicated a two-peak pattern characterized by a second period of growth in the stationary phase, comparing with the mutant. However, the mutant and the wild type strain exhibited similar capacity to form biofilm. In conclusion, the serp2169 gene could regulate bacterial growth in the stationary phase, and has no effect on biofilm formation.

Key words： Staphylococcus epidermidis homologous recombination serp2169

收稿日期: 2014-03-27

PACS:

R378.1

基金资助:皖南医学院博士科研启动基金(2011), 国家自然科学基金(No.81270091)

引用本文:

朱涛,赵艳丰,唐小牛,谷生丽,王少圣,李朝品. 表皮葡萄球菌serp2169基因敲除突变株的构建及其生长变化[J]. 中国人兽共患病学报, 2014, 30(9): 898-903. ZHU Tao,ZHAO Yan-feng,TANG Xiao-niu,GU Sheng-li,WANG Shao-sheng,LI Chao-pin. Construction of serp2169 knockout mutant in Staphylococcus epidermidis and characterization of its change in growth. Chinese Journal of Zoonoses, 2014, 30(9): 898-903.

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http://www.rsghb.cn/CN/ 或 http://www.rsghb.cn/CN/Y2014/V30/I9/898

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