Eukaryotic expression vector construction of outer membrane protein A from Acinetobacter baumannii and the incomplete autophagy in Hela cells induced by the protein
AN Zhiyuan
HUANG Xiaoxi
DING Wenyi
Abstract：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 LC3BII,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.
Keywords：Acinetobacter baumanniiOuter membrane protein AHela cells lineIncomplete autophagy
Publication Date：2017-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 996-1002 )