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

Eukaryotic expression vector construction of outer membrane protein A from Acinetobacter baumannii and the incomplete autophagy in Hela cells induced by the protein

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。

This study aims to construct the outer membrane protein A（OmpA） of Acinetobacter baumannii（A.baumannii） into an eukaryotic expression vector pEGFP-N1 and study the effect of OmpA on autophagy in Hela cells. The gene of OmpA of A. baumannii ATCC 19606 was amplified by PCR, then cloned into pEGFP-N1 vector.The recombinant eukaryotic expression vector pEGFP-OmpA was transfected into Hela cell lines through liposome transfection system. The OmpA expression in the Hela cells were detected by flow cytometry, Western blotting and immunofluorescence, while the localization of OmpA in Hela cells was detected by confocal microscopy. Western blot analysis and transmission electron microscopy were used to detect the autophagy of Hela cells elicited by OmpA. Double restriction enzymes digestion and DNA sequencing indicated that the recombinant eukaryotic expression vector pEGFP-OmpA was successfully constructed, while immunofluorescence assay showed that OmpA could be expressed abundantly in cytoplasm and nucleus of Hela cells. Overexpression of OmpA in Hela cells could increase the expression of autophagy-related proteins including LC3 BII, p62 and Beclin-1. Moreover, the autophagosome were found in Hela cells. OmpA could interfere p62 degradation and induce incomplete autophagy. In conclusion, the eukaryotic expression vector of pEGFP-OmpA have been constructed successfully,and can be expressed efficiently in Hela cells. Moreover, OmpA can trigger incomplete autophagy in Hela cells. This study lay a foundation for further research on the mechanism of autophagy induced by OmpA of Acinetobacter baumannii.