[Effective suppression of the infection and pathogenesis of simian immunodeficiency virus in transgenic lymphocytes mediated by self-cleavagble ribozyme].
OBJECTIVE
To obtain transgenic lymphocytes that are resistant to the infection and pathogenesis of simian immunodeficincy virus (SIV).


METHODS
A self-cleaving ribozyme was formed by introducing a fragment of ribozyme target sequence cleft by hammerhead ribozyme to the ribozyme downstream. Self-cleavable ribozyme gene was synthesized, amplified and cloned at the XhoI SalI site of pBluescript SK. The construct bearing 10 copies of ribozyme was obtained through successively cloning four times. The in vitro activity of self-cleavable ribozyme was tested, and then the self-cleaving ribozyme gene was transferred into mammalian expression vector pCI before transfection. The expression vectors harboring self-cleavable ribozyme gene were transfected into lymphocytes with liposome-mediated transfection method. The transfected cell clones were selected in the culturing medium containing 400 mg/L G418. The resistance of transgenic cell to simian immunodeficency virus infection was evaluated by immunofluorescence assays.


RESULTS
more than 85% of decameric ribozyme self-cleaved into monomeric ribozymes after incubation for 15 minutes at 37 degrees C. The number of cleavage products of decameric ribozyme during the first 25 minutes exceeds those of the monomeric ribozyme by 13.31% though the molar mass average of decameric ribozyme is less than 1/10 of that of the monomer ribozyme. The Northern dot blot results demonstrated that the monomer ribozyme gene and the decameric ribozyme gene had been transfected and expressed in the cells. SIV challenge trials showed that after 6 days after inoculation of SIV the cells transfected with the monomer ribozyme gene grew normally, no fused cell was observed; and most of the cells transfected with decameric gene grew well, but a few fused cells were observed; The inhibition rate was 100% for the cells transfected with monomer ribozyme gene to the SIV antigen positive cells, 91.2% for the cells transfected with decameric ribozyme gene, and 48% for the cells transfected with the empty expression vector.


CONCLUSIONS
The lymphocytes transfected with self-cleavable ribozyme gene make a strong resistance to SIV infection and pathogenesis. The cells transfected with monomeric ribozyme gene show a better inhibiting effect on SIV infection and pathogenesis in comparison with the cells transfected with decameric ribozyme gene.
Deng
W
W
Institute of Tropical Medicine, Guangzhou University of Traditional Chinese Medicine. Guangzhou 510407, China.
Zhang
F
F
Yang
X
X
D000818:Animals; D015152:Blotting, Northern; D015316:Genetic Therapy; D008214:Lymphocytes / Q000821:virology*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D016097:Simian Acquired Immunodeficiency Syndrome / Q000517:prevention & control*; D015302:Simian Immunodeficiency Virus / Q000472:pathogenicity*; D014162:Transfection
D016337:RNA, Catalytic