鲍曼不动杆菌外膜蛋白A真核表达载体构建及其诱导Hela细胞不完全自噬

发布时间：2017-12-30 22:30

本文关键词：鲍曼不动杆菌外膜蛋白A真核表达载体构建及其诱导Hela细胞不完全自噬　出处：《免疫学杂志》2017年11期 　论文类型：期刊论文

【摘要】：目的构建鲍曼不动杆菌ATCC19606(Acinetobacter baumannii ATCC 19606)外膜蛋白A(outer membrane protein A,OmpA)的真核表达载体pEGFP-OmpA,研究OmpA对Hela细胞自噬的影响。方法采用PCR方法扩增鲍曼不动杆菌ATCC19606的OmpA基因,将其克隆至真核表达载体pEGFP-N1,以构建重组质粒pEGFP-OmpA;用脂质体转染法将其转染Hela细胞;流式细胞术、细胞免疫荧光和Western blot检测OmpA表达水平;激光共聚焦显微镜检测OmpA在Hela细胞中的定位;Western blot及透射电子显微镜检测OmpA对Hela细胞自噬的影响。结果经双酶切及测序鉴定表明pEGFP-OmpA质粒构建成功;OmpA可以大量表达于Hela细胞的细胞质和细胞核中;OmpA可导致Hela细胞中自噬相关蛋白LC3BⅡ、p62和Beclin-1表达升高,而且在Hela细胞中发现自噬小体;OmpA会干扰p62降解,引发不完全自噬。结论本研究成功构建pEGFP-OmpA真核表达载体,它可以在Hela细胞中高效表达,并可导致Hela细胞不完全自噬,这为将来进一步研究鲍曼不动杆菌OmpA引起自噬的机制奠定基础。
[Abstract]:Objective to construct Bauman Acinetobacter ATCC19606 (Acinetobacter baumannii ATCC A (19606) outer membrane protein outer membrane protein A, OmpA) the eukaryotic expression vector pEGFP-OmpA. The effects of OmpA on autophagy in Hela cells. Methods OmpA gene amplification Bauman real coli ATCC19606 by PCR method, then cloned into eukaryotic expression vector pEGFP-N1. To construct the recombinant plasmid pEGFP-OmpA; liposome transfected into Hela cells; flow cytometry, cell immunofluorescence and Western blot detected the expression level of OmpA; detection OmpA laser scanning confocal microscope in Hela cells; OmpA Western blot influence the detection and transmission electron microscopy of autophagy in Hela cells. Results the double enzyme digestion and sequencing showed that the pEGFP-OmpA plasmid was successfully constructed; OmpA can be expressed in Hela cell cytoplasm and nucleus; OmpA can induce Hela cell autophagy. Protein LC3B II, p62 and Beclin-1 expression increased, and that of autophagosomes in Hela cells; OmpA may interfere with the degradation of p62, caused by incomplete autophagy. Conclusion this study has successfully constructed the eukaryotic expression vector pEGFP-OmpA, which can be efficiently expressed in Hela cells, and Hela cells can lead to incomplete autophagy, which for the future further study of Bauman Acinetobacter OmpA autophagy mechanism caused by the foundation.

【作者单位】：首都医科大学附属北京朝阳医院医学研究中心;中国医学科学院北京协和医院检验科;
【分类号】：R378
【正文快照】： 鲍曼不动杆菌是一种非发酵的革兰氏阴性条件致病菌,它通常感染免疫力低下的人群,可以导致肺炎、菌血症、泌尿系感染、脑膜炎等感染性疾病[1]。近年来,由于抗生素的滥用,产生了耐碳青霉烯的鲍曼不动杆菌,甚至出现了泛耐药和全耐药鲍曼不动杆菌,这给临床治疗带来了极大困难[2]。

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