铜绿假单胞菌青霉素结合蛋白-1b与藻酸盐产生关系的探讨

发布时间：2018-03-31 21:46

  本文选题：铜绿假单胞菌　切入点：生物被膜　出处：《华中科技大学》2012年硕士论文

【摘要】：【目的】利用RNA干扰技术（RNA interference，RNAi）抑制铜绿假单胞菌PBP-1b编码基因mrcB的表达，，探讨PBP-1b对藻酸盐编码基因algD产生的影响，以期为临床生物被膜耐药提供新的思路。 【方法】采用siRNA表达载体的方法，分别设计2对靶向mrcB基因shRNA序列，构建pMF54mrcB shRNA干扰载体；将构建好的干扰载体及空载分别电转入铜绿假单胞菌中，RT-PCR筛选抑制效率高的干扰载体，并分别计算第24小时、第三天、第六天的抑制率；同时用RT-PCR的方法分别检测PAO1标准株及转化株中藻酸盐编码基因algD分别在这三个时间点内的的表达情况，通过统计学处理观察algD的表达量有无差异。 【结果】经DNA测序鉴定，成功构建pMF54mrcB shRNA1/pMF54mrcB shRNA2干扰载体；将pMF54mrcB shRNA1/pMF54mrcB shRNA2/pMF54电转入铜绿假单胞菌中，分别与空白组对照，RT-PCR检测第24小时mrcB表达量pMF54mrcB shRNA2有明显统计学差异。计算其第24小时、第三天及第六天的抑制率分别为26%、41%、9%。单因素方差分析示标准株的algD基因在三个时间点表达的差异有统计学意义，第三天最高；转化株的表达量，在这三个时间点都比较高，无统计学意义。在这三个时间点用T-检验分别比较标准株及转化株algD基因的表达量，第三天无统计学意义，P＞0.05。 【结论】 PAO1标准株藻酸盐编码基因algD在生物被膜形成的第三天表达量最高；基因沉默的转化株其algD的表达量在这三个时间点明显增高，且其增高的时间提前，维持时间长；分别对标准株及转化株在三个时间点的algD表达量进行统计学处理，第三天表达量无统计学意义，可能与PAO1algD的表达量在第三天最高有关；PBP-1b与藻酸盐产生关系的具体调控机制还不详，有待于进一步研究发现。
[Abstract]:[objective] RNA interference technique was used to inhibit the expression of PBP-1b encoding gene mrcB in Pseudomonas aeruginosa, and to explore the effect of PBP-1b on the production of alginate encoding gene algD in order to provide a new idea for clinical biofilm drug resistance. [methods] using siRNA expression vector, two pairs of shRNA sequences targeting mrcB gene were designed to construct pMF54mrcB shRNA interference vector, and the constructed interference vector and no-load interference vector were respectively transferred into Pseudomonas aeruginosa by RT-PCR to screen the interference vectors with high inhibition efficiency. The inhibition rates of 24 hours, 3 days and 6 days were calculated, and the expression of alginate encoding gene algD in PAO1 standard strain and transformed strain were detected by RT-PCR. The expression of algD was observed by statistical analysis. [results] pMF54mrcB shRNA1/pMF54mrcB shRNA2 interference vector was successfully constructed by DNA sequencing, pMF54mrcB shRNA1/pMF54mrcB shRNA2/pMF54 was electrotransferred into Pseudomonas aeruginosa, and there was significant difference between pMF54mrcB shRNA1/pMF54mrcB shRNA2/pMF54 and control group in 24 h mrcB expression of pMF54mrcB shRNA2. The inhibition rates on the third day and the sixth day were 260.The single factor variance analysis showed that the algD gene expression of the standard strain was significantly different at the three time points, the highest on the third day; the expression level of the transformed strain was relatively high at these three time points. T- test was used to compare the expression of algD gene between the standard strain and the transformed strain at the three time points, but there was no significant difference between them on the third day (P > 0.05). [conclusion] the expression of alginate coding gene algD in PAO1 standard strain was the highest on the third day after the formation of biofilm, and the expression of algD in the transformed strain of gene silencing increased significantly at these three time points, and the time of increase was earlier and the time of maintenance was long. The algD expression of the standard strain and the transformed strain at three time points were statistically analyzed, but there was no statistical significance on the third day. The regulation mechanism of the relationship between PBP-1b and alginate production is unknown, which may be related to the highest expression of PAO1algD on the third day, which needs further study and discovery.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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