[Construction and verification of a novel triple-regulated oncolytic adenovirus carrying gene Pdcd5].
The purpose of this study was to construct a recombinant conditionally replicating adenovirus (CRAd) expressing programmed cell death 5 (pdcd5). Pdcd5 gene was inserted in the E3 region of SG600-a CRAd in which the key genes for virus replication E1a and E1b were controlled under the human telomerase reverse transcriptase promoter (hTERTp) and the hypoxia response element (HRE) respectively, and with a deletion of 24 nucleotides within CR2 region of E1a. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and polymerase chain reaction (PCR). The infection efficiencies of a recombined virus carrying enhanced green fluorescent protein (EGFP) in leukemic cell lines were observed by using fluorescence microscope. The relative pdcd5 expression levels of K562 after being infected with SG611-pdcd5 were detected by real-time quantitative PCR. The results showed that the construction of SG611-pdcd5 was completed and confirmed. Pdcd5, hTERTp, HRE, skeleton and fiber11 of recombinant adenovirus SG611-pdcd5 were successfully amplified. The infection efficiencies of SG611-EGFP were all above 70% in both leukemic K562 and MEG-01 cell lines. SG611-pdcd5 expressed pdcd5 with high efficiency in leukemic cells as compared with Ad-pdcd5 or SG611 (p < 0.001). The expression level of pdcd5 increased gradually along with the increase of MOI. It is concluded that the triple-regulated adenovirus of SG611-pdcd5 containing the pro-apopro-tic gene pdcd5 has been successfully established with high pdcd5 expression level in leukemic cells, indicating that the recombinant adenovirus, SG611-pdcd5, promises further development of targeted tumor gene therapy.
Xie
Min
M
Peking University People Hospital and Institute of Hematology, Beijing, China.
Wu
Hong-Ping
HP
Li
Lin-Fang
LF
Niu
Ji-Hong
JH
Chang
Yan
Y
Li
Jin-Lan
JL
Huang
Xiao-Jun
XJ
Ruan
Guo-Rui
GR
D000256:Adenoviridae / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D009363:Neoplasm Proteins / Q000235:genetics*; D050504:Oncolytic Viruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D019098:Telomerase / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D009363:Neoplasm Proteins; C117283:PDCD5 protein, human; C509186:TERT protein, human; D019098:Telomerase