贵州医科大学学报

2018, v.43;No.219(12) 1396-1401+1406

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高温对体外唾液链球菌clpP基因表达的影响
Effects of clpP Gene Expression at High Temperature in Streptococcus Salivarius in Vitro

朱铭慧;秦波;徐卫华;付雪飞;胡晓霞;
ZHU Minghui;QIN Bo;XU Weihua;FU Xuefei;HU Xiaoxia;Department of Orthodontics,the Affiliated Stomatological Hospital of Guizhou Medical University;Department of Physiology,School of Medical Science,Guizhou Medical University;

摘要(Abstract):

目的:探讨高温对体外培养的唾液链球菌(S. salivarius) clp P基因表达的影响。方法:取10例人唾液样本在55℃培养,挑选生成的菌落进行分离培养、16S r DNA扩增及DNA序列测定,采用BLAST分析及构建系统发育树,鉴定得到S. salivarius临床株;采用PCR方法对S. salivarius ATCC 13419模式株(阳性对照)和获得的S. salivarius临床株进行clp P基因的片段克隆及序列分析,再用实时荧光定量PCR方法比较S. salivarius在37℃及55℃下培养2 h时的clp P基因相对表达水平。结果:在55℃培养下,10例唾液样本中仅有2例有少量菌落生成,最终经分离鉴定得到2株S. salivarius临床株,分别命名为HS_01和HS_02;利用PCR方法克隆S.salivarius ATCC 13419模式株、HS_01及HS_02菌株的clp P基因片段,经实时荧光定量PCR方法检测显示,3个S. salivarius菌株在55℃条件下clp P基因的相对表达水平高于37℃培养时,差异有统计学意义(P <0. 05)。结论:高温刺激下获得的S. salivarius临床株的Clp P基因表达水平升高。
Objective: To detect the expression of clpP gene at high temperature in Streptococcus salivarius in vitro. Methods: Ten human saliva samples were cultured at 55 ℃ and colonies were selected for cultivation,isolation,16 S r DNA amplification and DNA sequencing. BLAST analysis and phylogenetic tree were used to identify S. salivarius clinical strains,which along with S. salivarius ATCC13419 type strains,were used for ClpP gene fragment cloning and sequencing by PCR method. And then real-time quantitative PCR method is applied to determine whether the strains have the difference or not in relative expressive amount of clpP gene while the temperature is at 37 ℃ and 55 ℃. Results:Our study gets two S. salivarius clinical strains-S. salivarius HS_01 and S. salivarius HS_02-under the temperature condition of 55 ℃. With PCR method,we cloned the partial clpP gene of S. salivarius ATCC 13419,S. salivarius HS_01 and S. salivarius HS_02. Real-time quantitative PCR showed that the relative expression levels of clpP gene in three S. salivarius strains at 55 ℃ were higher than those at 37 ℃. The difference was statistically significant( P < 0. 05). Conclusion: The expression level of ClpP gene in S. salivarius clinical strains obtained in high temperature stress is increased.

关键词(KeyWords): 高温;DNA序列测定;唾液链球菌;clpP基因;BLAST分析法;聚合酶链式反应;实时荧光定量PCR
high temperature;DNA sequencing;S.salivarius;clpP gene;BLAST analysis;polymerase chain reaction;real-time quantitative PCR

Abstract:

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基金项目(Foundation): 贵州省科技计划项目[黔科合LH字(2017)7201号]

作者(Author): 朱铭慧;秦波;徐卫华;付雪飞;胡晓霞;
ZHU Minghui;QIN Bo;XU Weihua;FU Xuefei;HU Xiaoxia;Department of Orthodontics,the Affiliated Stomatological Hospital of Guizhou Medical University;Department of Physiology,School of Medical Science,Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2018.12.007

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