靶向siRNA抑制金黃色葡萄球菌coa基因表達的研究
發(fā)布時間：2019-06-25 13:52
【摘要】：目的探討直接導入以金黃色葡萄球菌(SA) coa基因為靶標的siRNA后,SA的coa mRNA表達和葡萄球菌凝固酶(SC)蛋白水平是否受到抑制。 方法根據(jù)GenBank coa序列設計siRNA,采用體外化學合成法合成。繪制SA的時間-OD600曲線,判斷SA的對數(shù)生長期。在SA培養(yǎng)過程中,分別將靶向coa的siRNA、陰性對照siRNA和培養(yǎng)基直接加入siRNA組、陰性對照siRNA(NC)組和空白對照(Blank)組,分3次加入,每次50μ1(20gM)。在首次加入siRNA后60min(A組)、90min(B組)和120min(C組)3個時間點收集菌液,采用Trizol法提取總RNA和總蛋白。應用實時熒光定量PCR檢測3個時間點(A組60min、B組90min和C組120moin)的5組(1號siRNA、2號siRNA、3號siRNA、 NC和Blank)coa mRNA的表達。應用Western blot檢測2個時間點(A組60min和B組90min)的2組(1號siRNA和Blank) SC蛋白表達水平。 結果根據(jù)時間-OD600曲線,判斷OD600=0.420-1.499為SA的對數(shù)生長期,選擇OD600=0.012為試驗起點,2h后為終點。實時熒光定量PCR結果顯示,在A組和B組,1號siRNA組的coa mRNA的表達均低于NC組和Blank組(P0.05),且NC組和Blank組之間的差異無統(tǒng)計學意義。Western blot結果顯示,在A組和B組,與Blank組相比,1號siRNA組的SC蛋白的表達有所降低。 結論篩選出1對以SA的coa基因為靶標的siRNA (5'-CGCAUUAGCAGUUGC AUCUAGCUUATT-3',5'-UAAGCUAGAUGCAACUGCUAAUGCGTT-3')。采用少量多次的直接導入siRNA法,coa mRNA和SC蛋白的表達水平均有所降低。
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378
本文編號：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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378
【引證文獻】
相關碩士學位論文 前2條
1 馬瓊;養(yǎng)營活血湯對肝纖維化大鼠Wnt/β-catenin信號通路的影響[D];寧夏醫(yī)科大學;2018年
2 董小云;綿馬貫眾素對金黃色葡萄球菌vWbp抑制作用的研究[D];吉林大學;2017年
，本文編號：2505733
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