[Construction of the expression vectors of HDV ribozymes and their intracellular inhibiting activity against HCV RNA].
OBJECTIVE
To investigate whether HDV ribozymes can intracellularly inhibit HCV RNA.


METHODS
The mammalian expression vectors, pC1-RzC1, pC1-RzC2 and pC1-RzC3, containing ribozymes cDNA of RzC1, RzC2, and RzC3, were constructed targeting different HCV-5' NCR-C RNA regions. Then the HCV-positive fetal hepatocytes were transfected with these plasmids using liposome-mediated method. The inhibitory effects of HDV ribozymes were evaluated by HCV RNA quantitation in cultured cells and the supernatants.


RESULTS
(1) All the three HDV ribozymes were inserted into the expression vector. (2) Fetal hepatocytes were infected with HCV proven by RT-PCR and fluorescent quantitative PCR and expressed HCV NS3 and NS5 antigens by immunocytochemistry. (3) HDV ribozymes inhibited the activity of the target HCV RNA at expect positions in HCV-positive hepatocytes. At 0.5 micromol/L, the inhibitory rate of pC1-RzC1, pC1-RzC2, and pC1-RzC3 was 53.2%, 50.5 %, and 10.6% respectively. PC1-RzC1 was used continuously for one week, showing the inhibitory rate of 60.7%, 64.2%, 68.4%, 71.9%, 78.8% and 83.1% on the 2nd, 3rd, 4th, 5th, 6th and 7th day.


CONCLUSIONS
The inhibitory activity of pC1-RzC1 (107-113nt) and pC1-RzC2 (268-274nt) is greater than that of pC1-RzC3 (345-351nt) in HCV-positive hepatocytes.
Guo
Huan-zhen
HZ
Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Mao
Qing
Q
Li
Qi-fen
QF
Wang
Yu-ming
YM
Yu
Le-cheng
LC
Jiang
Ye-gui
YG
D015316:Genetic Therapy; D005822:Genetic Vectors; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics; D006526:Hepatitis C / Q000188:drug therapy*; D003698:Hepatitis Delta Virus / Q000235:genetics*; D010957:Plasmids; D016337:RNA, Catalytic / Q000627:therapeutic use*; D012367:RNA, Viral / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016337:RNA, Catalytic; D012367:RNA, Viral