[Synthesis and preliminary study on cleavage activity of ribozyme to hepatitis B virus preS2 gene in vitro].
In this paper, a multi-target hammerhead ribozyme gene was synthesized directed against 110, 122 and 132 sites of nucleotide of HBV preS2 gene. The target gene fragment was cut from HBV genome containing plasmid pCP10. Both of the ribozyme and the target gene fragments were cloned into pGEM3Zf(-) plasmid and sequenced by dideoxy chain termination method. The transcription of both gene fragments was performed in vitro utilizing T7 RNA promoter in pGEM3Zf (-) plasmid. The cleavage activity of ribozyme to substrate was confirmed in vitro. For further evaluating intracellular function of ribozyme, two ribozyme-retroviral recombinant plasmids with different promoter type pDOR-ripe and tRNA-ripe were constructed. Pseudo-virus was collected through routine packaging procedure and transduted into 2.2.15 cells. RIA data showed a stable inhibition of pHSA-R antigen expression to the lowest extent of 41.01% +/- 4.16 and the highest extent of 51.45% +/- 4.57 within 4 weeks after transduction. No influence, however, on HBsAg and HBeAg expression was demonstrated after ribozyme gene transfer.
Xu
D
D
Biological Engineering Laboratory of 302 Hospital, Beijing 100039.
Han
F
F
Shi
H
H
D003001:Cloning, Molecular; D053938:DNA Fragmentation; D005814:Genes, Viral; D015316:Genetic Therapy; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000201:enzymology*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*
D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D011498:Protein Precursors; D016337:RNA, Catalytic; C050807:presurface protein 2, hepatitis B surface antigen