靶向siRNA抑制金黄色葡萄球菌coa基因表达的研究
发布时间：2019-06-25 13:52
【摘要】：目的探讨直接导入以金黄色葡萄球菌(SA) coa基因为靶标的siRNA后,SA的coa mRNA表达和葡萄球菌凝固酶(SC)蛋白水平是否受到抑制。 方法根据GenBank coa序列设计siRNA,采用体外化学合成法合成。绘制SA的时间-OD600曲线,判断SA的对数生长期。在SA培养过程中,分别将靶向coa的siRNA、阴性对照siRNA和培养基直接加入siRNA组、阴性对照siRNA(NC)组和空白对照(Blank)组,分3次加入,每次50μ1(20gM)。在首次加入siRNA后60min(A组)、90min(B组)和120min(C组)3个时间点收集菌液,采用Trizol法提取总RNA和总蛋白。应用实时荧光定量PCR检测3个时间点(A组60min、B组90min和C组120moin)的5组(1号siRNA、2号siRNA、3号siRNA、 NC和Blank)coa mRNA的表达。应用Western blot检测2个时间点(A组60min和B组90min)的2组(1号siRNA和Blank) SC蛋白表达水平。 结果根据时间-OD600曲线,判断OD600=0.420-1.499为SA的对数生长期,选择OD600=0.012为试验起点,2h后为终点。实时荧光定量PCR结果显示,在A组和B组,1号siRNA组的coa mRNA的表达均低于NC组和Blank组(P0.05),且NC组和Blank组之间的差异无统计学意义。Western blot结果显示,在A组和B组,与Blank组相比,1号siRNA组的SC蛋白的表达有所降低。 结论筛选出1对以SA的coa基因为靶标的siRNA (5'-CGCAUUAGCAGUUGC AUCUAGCUUATT-3',5'-UAAGCUAGAUGCAACUGCUAAUGCGTT-3')。采用少量多次的直接导入siRNA法,coa mRNA和SC蛋白的表达水平均有所降低。
[Abstract]:Objective To investigate whether the expression of coa mRNA and the level of coagulase (SC) protein of SA after direct introduction of siRNAs targeting S. aureus (SA) coa gene were inhibited. Methods The siRNA was designed according to the GenBank coa sequence and synthesized by in vitro chemical synthesis. So as to draw the time-OD600 curve of SA to judge the logarithmic growth of SA. During the SA culture, siRNA, negative control siRNA and culture medium of the target coa were added directly to the siRNA group, the negative control siRNA (NC) group and the blank control (Blank) group, and the siRNA group, the negative control siRNA (NC) group and the blank control (Blank) group were directly added, and the siRNA group and the negative control siRNA (20 gM) were added each time. The total RNA and total egg were extracted by Trizol method at the time of 60 min (group A),90 min (B group) and 120 min (C group) after the first addition of siRNA. White. Table of 5 groups (No.1 siRNA, No.2 siRNA, No.3 siRNA, NC and Blank) coa mRNA were detected by real-time fluorescence quantitative PCR for 3 time points (60 min in group A,90 min in group B and 120 moin in group C). D. Two groups (No.1 siRNA and Blank) SC protein expression water were detected by Western blot for 2 time points (60 min in group A and 90 min in group B). The result is determined according to the time-OD600 curve, OD600 = 0.420-1.499 is the logarithmic growth phase of SA, OD600 = 0.012 is selected as the starting point of the test, and after 2 h, The results of real-time fluorescence quantitative PCR showed that the expression of coa mRNA in group A and group B was lower than that of NC group and Blank group (P0.05), and there was no statistical difference between NC group and Blank group. The results of Western blot showed that in group A and group B, the expression of SC protein in No.1 siRNA group was compared with that of Blank group. Conclusion:1 siRNA (5 '-CGCAUAGCAGUUGC AUCUCUUATT-3',5 '-UAAGCUAGAUGCUAAUGCGTT-3') with a SA-based coa gene was selected. The expression level of coa mRNA and SC protein was directly introduced into the siRNA method with less amount of the siRNA.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
本文编号：2505733
[Abstract]:Objective To investigate whether the expression of coa mRNA and the level of coagulase (SC) protein of SA after direct introduction of siRNAs targeting S. aureus (SA) coa gene were inhibited. Methods The siRNA was designed according to the GenBank coa sequence and synthesized by in vitro chemical synthesis. So as to draw the time-OD600 curve of SA to judge the logarithmic growth of SA. During the SA culture, siRNA, negative control siRNA and culture medium of the target coa were added directly to the siRNA group, the negative control siRNA (NC) group and the blank control (Blank) group, and the siRNA group, the negative control siRNA (NC) group and the blank control (Blank) group were directly added, and the siRNA group and the negative control siRNA (20 gM) were added each time. The total RNA and total egg were extracted by Trizol method at the time of 60 min (group A),90 min (B group) and 120 min (C group) after the first addition of siRNA. White. Table of 5 groups (No.1 siRNA, No.2 siRNA, No.3 siRNA, NC and Blank) coa mRNA were detected by real-time fluorescence quantitative PCR for 3 time points (60 min in group A,90 min in group B and 120 moin in group C). D. Two groups (No.1 siRNA and Blank) SC protein expression water were detected by Western blot for 2 time points (60 min in group A and 90 min in group B). The result is determined according to the time-OD600 curve, OD600 = 0.420-1.499 is the logarithmic growth phase of SA, OD600 = 0.012 is selected as the starting point of the test, and after 2 h, The results of real-time fluorescence quantitative PCR showed that the expression of coa mRNA in group A and group B was lower than that of NC group and Blank group (P0.05), and there was no statistical difference between NC group and Blank group. The results of Western blot showed that in group A and group B, the expression of SC protein in No.1 siRNA group was compared with that of Blank group. Conclusion:1 siRNA (5 '-CGCAUAGCAGUUGC AUCUCUUATT-3',5 '-UAAGCUAGAUGCUAAUGCGTT-3') with a SA-based coa gene was selected. The expression level of coa mRNA and SC protein was directly introduced into the siRNA method with less amount of the siRNA.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
【引证文献】
相关硕士学位论文 前2条
1 马琼;养营活血汤对肝纤维化大鼠Wnt/β-catenin信号通路的影响[D];宁夏医科大学;2018年
2 董小云;绵马贯众素对金黄色葡萄球菌vWbp抑制作用的研究[D];吉林大学;2017年
，本文编号：2505733
本文链接：https://www.wllwen.com/xiyixuelunwen/2505733.html
最近更新
教材专著
