Construction and functional analysis of a lentiviral expression vector containing a scavenger receptor (SR-PSOX) that binds uniquely phosphatidylserine and oxidized lipoprotein.
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 non-dividing 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 SR-PSOX, 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)-alpha 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-alpha 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-alpha.
Quan
Zhihua
Z
Institute of Cardiovascular Disease, Nanhua University, Hengyang, China.
Yang
Huiling
H
Yang
Yongzong
Y
Yan
Bin
B
Cao
Renxian
R
Wen
Gebo
G
Liu
Changhui
C
Xu
Yangyan
Y
D050197:Atherosclerosis / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D001483:Base Sequence; D002460:Cell Line; D000075743:Chemokine CXCL16; D019743:Chemokines, CXC / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005487:Foam Cells / Q000378:metabolism / Q000473:pathology; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008969:Molecular Sequence Data; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding; D051116:Receptors, Scavenger / Q000235:genetics* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
C423727:CXCL16 protein, human; D000075743:Chemokine CXCL16; D019743:Chemokines, CXC; D017931:DNA Primers; D008077:Lipoproteins, LDL; D010718:Phosphatidylserines; D051116:Receptors, Scavenger; D014409:Tumor Necrosis Factor-alpha; C099252:oxidized low density lipoprotein