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通过反义寡核苷酸-核酶复合物沉默临床铜绿假单胞菌分离株中的 acpP 基因。

Silencing acpP gene via antisense oligonucleotide-niosome complex in clinical Pseudomonas aeruginosa isolates.

机构信息

Izmir Kâtip Celebi University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Izmir, Turkey.

Akdeniz University, Faculty of Medicine, Department of Pharmacology, Antalya, Turkey; Akdeniz University, Health Sciences Institute, Department of Medical Biotechnology, Antalya, Turkey; Akdeniz University, Health Sciences Institute, Department of Gene and Cell Therapy, Antalya, Turkey.

出版信息

Res Microbiol. 2021 Jun-Aug;172(4-5):103834. doi: 10.1016/j.resmic.2021.103834. Epub 2021 Apr 21.

Abstract

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is one of the major causes of nosocomial infections. In addition to its physiological adaptation capacity, it can develop resistance to disinfectants and antibiotics through various mechanisms. Recently, new eradication methods are gaining attention. Therefore, in this study, an LNA-2'-O-methyl hybrid antisense oligonucleotide targeting the acyl carrier protein P (acpP) gene was introduced into P. aeruginosa isolates. The design was determined through sequence analysis and prediction of the secondary structure of mRNA by software. Niosomes were used for enhancing cellular uptake. The control of the binding and transfection ability of the sequence was determined fluorometrically by labeling with 6-Fam. The effects were determined with broth microdilution method and qPCR studies. Eight different formulations were prepared. Among these, one formulation has shown to have ASO complexation ability whose composition was 312 μl Span 80 + 69.5 mg Cholesterol+ 36.4 mg CTAB+1 ml Chloroform and 5 ml dHO. Thus this formulation was determined as the delivery system for the next stages. Significant gene inhibition was detected at the six isolates. Results of this study suggested that niosomes can be used as a delivery system for cellular uptake of ASO and could eliminate bacterial growth.

摘要

铜绿假单胞菌是一种机会性革兰氏阴性病原体,是医院感染的主要原因之一。除了其生理适应能力外,它还可以通过各种机制对消毒剂和抗生素产生耐药性。最近,新的根除方法引起了人们的关注。因此,在这项研究中,引入了一种针对酰基辅酶 A 蛋白 P (acpP) 基因的 LNA-2'-O-甲基杂交反义寡核苷酸到铜绿假单胞菌分离株中。设计是通过序列分析和软件预测 mRNA 的二级结构确定的。使用奈脂质体来增强细胞摄取。通过用 6-Fam 标记来荧光测定序列的结合和转染能力的对照。通过肉汤微量稀释法和 qPCR 研究来确定效果。制备了八种不同的配方。其中,一种制剂具有 ASO 复合物形成能力,其组成为 312 μl Span 80 + 69.5 mg 胆固醇+ 36.4 mg CTAB + 1 ml 氯仿和 5 ml dHO。因此,该制剂被确定为下一阶段的递送系统。在六个分离株中检测到了显著的基因抑制。这项研究的结果表明,奈脂质体可用作 ASO 细胞摄取的递送系统,并可以消除细菌生长。

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