变形链球菌LuxS基因缺陷株生物学性状的初步研究

发布时间：2018-06-11 18:25

本文选题：变形链球菌 + 缺陷株　；参考：《重庆医科大学》2011年硕士论文

【摘要】：【目的】LuxS基因是变形链球菌生物膜早期形成过程中的关键基因,为探讨变形链球菌LuxS基因在生物膜形成中的调控机制,本研究拟构建该基因的缺陷株,并对该基因缺失后变形链球菌相关生物学性状的改变进行初步探讨。【材料和方法】(1)采用长臂同源多聚酶链反应(LFH-PCR)方法构建含红霉素耐药基因片段的LuxS基因上、下游同源序列的连接片段,转化到变形链球菌中,在红霉素的平板上筛选缺陷株,并进行PCR及序列鉴定。(2)将变形链球菌标准菌和构建获得的LuxS基因缺陷株分别在BHI液体培养基、含2%葡萄糖的BHI液体培养基、含2%蔗糖的BHI液体培养基3个营养环境中生长,采用722S型可见光分光光度计,对细菌生长曲线的变化进行测定。(3)将变形链球菌LuxS基因缺陷株和标准菌在BHI液体培养基,含2%葡萄糖的BHI液体培养基,含2%蔗糖的BHI液体培养基3个营养环境中培养24小时,通过扫描电镜对不同营养环境中标准菌及LuxS基因缺陷株早期生物膜的形成差异进行比较。(4)将处于对数生长期的标准菌及缺陷株分别接种于含2%蔗糖的BHI液体培养基中形成细菌生物膜,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术,初步分析生物膜形成初期不同时间点的可溶性蛋白表达情况。【结果】(1)经PCR和DNA序列测定分析证实变形链球菌LuxS基因缺陷菌株构建成功。(2)在3种不同的营养环境中,变形链球菌UA159LuxS基因缺陷株的生长速度较变形链球菌UA159标准菌株相对略快。(3)变形链球菌LuxS基因缺陷株在富含蔗糖的环境中,生物膜形成能力减弱,且形成的生物膜相对疏松;而在富含葡萄糖以及不含糖的环境中,缺陷株与标准菌形成生物膜的能力差异不大。(4)对5个时间点上变形链球菌标准菌和LuxS缺陷株生物膜中菌体可溶性蛋白未见特异表达的蛋白条带。【结论】(1)本研究成功构建出变形链球菌LuxS基因缺陷株,为后期针对变形链球菌LuxS基因的相关研究奠定基础。(2)LuxS基因缺陷后会对变形链球菌的生长能力产生影响,当环境中营养增加时,标准菌和缺陷株数量的最高峰值增加,并且峰值接近。(3)LuxS基因缺陷会对变型链球菌生物膜的形成产生影响,并且LuxS基因对变形链球菌生物膜的调控是蔗糖依赖型的。(4)在本实验条件下使用SDS-PAGE技术,结果表明,LuxS基因缺陷后,其早期生物膜形成过程中可溶性蛋白的表达量与标准菌之间未检出明显差异。
[Abstract]:[objective] LuxS gene is a key gene in the early biofilm formation of Streptococcus mutans. The changes of related biological characters of Streptococcus mutans after the deletion of the gene were preliminarily discussed. [materials and methods] using long arm homologous polymerase chain reaction (LFH-PCR) method, the LuxS gene containing erythromycin resistance gene fragment was constructed. The downstream homologous sequence was transformed into Streptococcus mutans, and the defective strains were screened on the erythromycin plate. PCR and sequence identification were performed. The standard strain of Streptococcus mutans and the LuxS gene deficient strain were constructed in BHI liquid medium, respectively. BHI liquid medium containing 2% glucose and BHI liquid medium containing 2% sucrose were grown in three nutrient environments. The strain and standard strain of Streptococcus mutans LuxS gene were cultured in BHI liquid medium, BHI liquid medium containing 2% glucose and BHI liquid medium containing 2% sucrose for 24 hours. The differences in the formation of early biofilm between standard bacteria and LuxS gene deficient strains in different nutrient environments were compared by scanning electron microscope (SEM). The standard bacteria and defective strains in logarithmic growth period were inoculated into BHI liquid culture containing 2% sucrose. Forming bacterial biofilm in the base, Sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE) technique was used. The expression of soluble protein at different time points in the early stage of biofilm formation was preliminarily analyzed. [results] the PCR and DNA sequencing analysis showed that Streptococcus mutans LuxS gene deficient strain was successfully constructed. The growth rate of Streptococcus mutans UA159LuxS gene deficiency strain was a little faster than that of Streptococcus mutans UA159 standard strain.) Streptococcus mutans LuxS gene defect strain decreased its biofilm formation ability in sucrose rich environment, and the biofilm formation was relatively loose. And in glucose-rich and sugar-free environments, The ability of biofilm formation between the defective strain and the standard strain was not different. 4) there were no specific expression bands of soluble protein in the biofilm of Streptococcus mutans and LuxS deficient strain at 5 time points. [conclusion] 1) The LuxS gene deficient strain of Streptococcus mutans was successfully constructed. For the later study of LuxS gene of Streptococcus mutans, the deficiency of LuxS gene will affect the growth ability of Streptococcus mutans. When the nutrition in the environment increases, the peak number of standard bacteria and defective strains increases. The peak value of LuxS gene was close to that of Streptococcus mutans biofilm formation, and the regulation of LuxS gene on Streptococcus mutans biofilm was sucrose dependent. The results showed that there was no significant difference between the expression of soluble protein in the early biofilm formation and the standard bacteria after LuxS gene deficiency.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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