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目的:利用反义策略,设计、筛选针对agrA mRNA的反义锁核酸,阻断agr群体感应系统,降低MRSA毒力因子的表达,减小细菌生存压力,为抗耐药菌感染提供一种新策略。方法:应用Primer Premier 5.0和RNA structure 4.5两种软件,设计、筛选2条针对agrA mRNA的反义寡核苷酸序列,对其进行锁核酸修饰,并与透膜肽(KFF)3K共价连接,将这两条反义序列导入细菌体内。体外与MRSA共培养,观察细菌生存情况。实时定量PCR检测其对agrA及agr群体感受系统的效应分子RNAⅢ和下游毒力基因hla的转录水平的影响,Western blot检测其是否能抑制α-溶血素的表达。结果:两条反义锁核酸序列PLNA34和PLNA522在体外均无抗菌活性,但都能不同程度的抑制agrA,RNAⅢ和hla的转录水平;相比较而言,PLNA34的抑制效果更佳,并能明显抑制α-溶血素的分泌表达。结论:agrA可作为抗MRSA感染的新靶点,为新型抗MRSA药物的研发提供了理论基础。
OBJECTIVE: To design and screen the antisense-locked nucleic acid against agrA mRNA by antisense strategy, block the induction system of agr population, reduce the expression of virulence factor of MRSA, reduce the pressure of bacterial survival and provide a new anti-drug-resistant bacterial infection Strategy. METHODS: Two antisense oligonucleotide sequences targeting agrA mRNA were designed and screened by Primer Premier 5.0 and RNA structure 4.5 software. The antisense oligonucleotides were designed to be locked with nucleic acids and covalently linked to KFF 3K , The two antisense sequences into bacteria. In vitro and MRSA co-culture, observation of bacterial survival. Real-time quantitative PCR was used to detect the transcriptional level of effector RNAⅢ and downstream virulence gene hla in agrA and agr population. Western blot was used to detect the expression of α-hemolysin. RESULTS: The two antisense-locked nucleic acid sequences, PLNA34 and PLNA522, showed no antibacterial activity in vitro, but all of them inhibited the transcription of agrA, RNAⅢ and hla to some extent. Compared with PLNA34, PLNA34 showed better inhibitory effect Secretion of α-hemolysin is inhibited. Conclusion: agrA can be used as a new target for anti-MRSA infection and provide a theoretical basis for the development of new anti-MRSA drugs.