[Construction of RNA-containing virus-like nanoparticles expression vector with cysteine residues on surface and fluorescent decoration].
Site-directed mutagenesis was performed at the codon 15 of the MS2 bacteriophage coat protein gene,which had been cloned to the virus-like particles expression vector containing non-self RNA fragment. The produced expression vector,termed pARSC, was transformed to E. coli DH5alpha. The positive clones were selected and proliferated. The harvested cells were treated with sonication and the supernatant was then subjected to linear sucrose density gradients centrifugation (15% to 60%) at 32000 r/min for 4 h at 4 degrees C. The virus-like particles, VLP-Cy, were collected at 35% sucrose density. The particles were examined by transmission electron microscopy and the spherical viral particles of approximately 27 nm in diameter were found. The thiolated VLP-Cy was then chemically modified with fluorescein -5'-maleimide. The covalent fluorescent labeling was confirmed by absorption analysis, SDS-PAGE and MALDI-TOF mass spectroscopy. This is the first report of preparation of RNA-containing natural fluorescent nanoparticles. The study highlight the versatility of MS2 bacteriophage capsids as building blocks for functional nanomaterials construction for a variety of application purposes.
Cheng
Yang-Jian
YJ
The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen 361005, China. yjcheng11@163.com
Liang
Ji-Xuan
JX
Li
Qing-Ge
QG
D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D003062:Codon / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D017909:Levivirus / Q000235:genetics* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D016297:Mutagenesis, Site-Directed; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D012367:RNA, Viral / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D036022:Capsid Proteins; D003062:Codon; D005452:Fluoresceins; D012367:RNA, Viral; D011994:Recombinant Proteins; C037041:fluorescein 5-maleimide; D003545:Cysteine