Anti-HBV activity of TRL mediated by recombinant adenovirus.
OBJECTIVE
To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication.


METHODS
Based on previously constructed pcDNA3.1(-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGloxdeltaE1,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.


RESULTS
RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63+/-0.14 vs 1.60+/-0.47, P = 0.0266, <0.05) and other control groups (0.63+/-0.14 vs 8.50+/-2.78, 8.25+/-2.26, 8.25+/-2.29, 8.50+/-1.51, 8.57+/-1.63, P<0.01). MTT assay suggested that there were no significant differences in cell metabolic activity between groups (P>0.05).


CONCLUSIONS
The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.
Gong
Wei-Dong
WD
Department of Oncology, Zhujiang Hospital, Nanfang Medical University, Guangzhou 510280, Guangdong Province, China. gongwd70@fmmu.edu
Zhao
Ya
Y
Yi
Jun
J
Ding
Jin
J
Liu
Jun
J
Xue
Cai-Fang
CF
D000256:Adenoviridae / Q000235:genetics*; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D015316:Genetic Therapy / Q000379:methods*; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development*; D006801:Humans; D008113:Liver Neoplasms; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014779:Virus Replication
D011994:Recombinant Proteins