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金黄色葡萄球菌酚溶调控蛋白α小肽敲除对自溶素AtlA蛋白表达的影响
Effect of PSMα knockout on autolysin AtlA expression in
Staphylococcus aureus - 第三军医大学学报 2017年39卷第21期 页码：2066-2071
- 作者机构：
400038 重庆，第三军医大学基础医学部微生物学教研室
- 作者简介：
[黎庶，E-mail: lishu72@aliyun.comLI Shu, E-mail: lishu72@aliyun.com
- 基金信息：国家自然科学基金(31470241);国家自然科学基金(31570127);国家级大学生创新创业训练计划(201690031002)
DOI：10.16016/j.1000-5404.201706121
中图分类号： R378.11;R394-33;R394.3收稿：2017-06-21，
修回：2017-07-21，
纸质出版：2017-11-15
- 稿件说明：
移动端阅览
谭利, 李思瑞, 姜北, 等. 金黄色葡萄球菌酚溶调控蛋白α小肽敲除对自溶素AtlA蛋白表达的影响[J]. 第三军医大学学报, 2017,39(21):2066-2071.
Li TAN, Sirui LI, Bei JIANG, et al. Effect of PSMα knockout on autolysin AtlA expression in
Staphylococcus aureus [J]. Journal of Third Military Medical University, 2017, 39(21): 2066-2071.谭利, 李思瑞, 姜北, 等. 金黄色葡萄球菌酚溶调控蛋白α小肽敲除对自溶素AtlA蛋白表达的影响[J]. 第三军医大学学报, 2017,39(21):2066-2071. DOI： 10.16016/j.1000-5404.201706121.
Li TAN, Sirui LI, Bei JIANG, et al. Effect of PSMα knockout on autolysin AtlA expression in
Staphylococcus aureus [J]. Journal of Third Military Medical University, 2017, 39(21): 2066-2071. DOI： 10.16016/j.1000-5404.201706121.
摘要
目的
2
利用同源重组技术构建金黄色葡萄球菌Newman psmα基因缺失突变株，探究psmα缺失对金黄色葡萄球菌蛋白表达的影响。
方法
2
分别扩增psmα基因的上下游同源臂，根据同源重组原理，构建pBT2基因打靶载体，利用同源重组技术，构建psmα缺失的Newman突变株。并对野生株和突变株培养上清蛋白进行研究。
结果
2
突变株培养上清中一个相对分子质量约为135×10
3
的蛋白表达水平较野生株明显减少甚至缺失；质谱分析提示该蛋白为金黄色葡萄球菌atlA基因编码的双功能自溶素前体蛋白(AtlA蛋白)；实时荧光定量PCR结果显示psmα基因敲除株atlA基因的转录水平较野生株显著降低。
结论
2
psmα缺失后能够降低atlA基因的转录表达水平。
Abstract
Objective
2
To construct the phenol-soluble modulin (psmα) locus gene knockout mutant of
Staphylococcus aureus
(
S. aureus
) and evaluate the effect of PSMα peptides on the expression of secretory proteins.
Methods
2
The temperature-sensitive
S. aureus
knock-out vectors were constructed after 2 homologous sequences around the psmα locus were cloned and inserted into pBT2 vector to construct a knockout plasmid pBT2∷psmα. Then the obtained plasmids pBT2∷psmα were transferred into
S. aureus
strain Newman
and the Newman∷Δpsmα mutant were prepared with homologous recombination. The proteins in the culture supernatants of Newman wild type strain and Newman∷Δpsmα mutant were analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. The transcriptional level of altA gene was detected using quantitative real-time PCR.
Results
2
SDS-PAGE confirmed that there was a band missing at the relative mass weight of 135×10
3
when compared with the results of the wildtype strain. Mass spectrometry indicated that the protein was dual functionality autolyzed precursor protein (AtlA) in Newman∷Δpsmα mutant. RT-PCR also showed the atlA gene transcription was significantly reduced in the psmα mutant strain.
Conclusion
2
The PSMα knockout in S. aureus reduces the expression of atlA at transcriptional level.
关键词
Keywords
references
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