Construction and Functional Analysis of a Lentiviral Expression Vector Containing a Scavenger Receptor (SR-PSOX) that Binds Uniquely Phosphatidylserine and Oxidized Lipoprotein

摘要

The aim of this study is to construct a lentiviral expression vector containing a scavenger receptor (SR-PSOX) that binds with uniquely phosphatidylserine and oxidized lipoprotein with six histidine tags and to investigate the function of SR-PSOX in atherosclerosis.We utilize the ViraPower lentiviral expression system which was efficient to deliver in vitro or in vivo the target gene into dividing and nondividing mammalian cells using an enhanced biosafety replication-incompetent lentivirus.The blunt-end sequence was amplified using the reverse transcription-polymerase chain reaction and directional TOPO cloning reaction.Through a pair of the cytomegalovirus forward primer and the reverse primer of SRPSOX,the correct clones were identified by polymerase chain reaction and sequencing.The ViraPower packaging mix and SR-PSOX-pLenti6/V5 TOPO expression plasmid were co-transfected into the 293FT cell line using Lipofectamine 2000.The expression of endogenous and exogenous SR-PSOX as well as tumor necrosis factor (TNF)-α protein in various foam cell models at different time points were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,Western blot and indirect immunofluorescence assay.Western blot and immunofluorescence analysis confirmed that the expressions of SR-PSOX and TNF-α protein were upregulated in foam cell models.Our data suggested that the overexpression of recombinant human SR-PSOX protein can promote foam cell formation and upregulate the expression of the inflammatory factor TNF-α.