[Preparation of Recombinant Human Adenoviruses Labeled with miniSOG].
We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.
Xiaohui
X
Zou
Rong
R
Xiao
Xiaojuan
X
Guo
Jianguo
J
Qu
Zhuozhuang
Z
Lu
Tao
T
Hong
D000260:Adenoviruses, Human / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D056949:Phototropins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026082:Singlet Oxygen / Q000737:chemistry; D013194:Staining and Labeling; D014162:Transfection
D029681:Arabidopsis Proteins; D005420:Flavoproteins; D056949:Phototropins; D026082:Singlet Oxygen