Effect On Telomerase Activity In Giardia Canis By GCV Vector-mediated Miniature Ribozyme

It is important that telomerase expression during proliferation of eukaryotic cell growth, and it has been identified that it could impair the proliferation of eucaryotic cell by suppression of telomerase activity with the specific inhibitors of telomerase. Therefore, studying the activity of telomerase will provide a new method to treatment parasitic disease. In this study,Giardia Canis virus universial transfer vector was constructed and enhance green fluorescent protein was successfuLly expressed in the Giardia canis. Telomeric repetitive DNA sequences of Giardia Canis was identificated. Giardia Canis telomerase activity was detected and we confirmed the high telomerase activity presented in trophozoites not in cysts. Two miniature ribozyme mosaicism Giardia canis virus transfer vector TRzS and TRzL were firstly constructed and the cleavage activity of Giardia canis virus﹣mediated Miniature ribozyme for GcTERT in vitro transcript and the effect on inhibition of GcTERT gene expression in Giardia canis by GCV transfer vector -mediated Miniature ribozyme were studied. This study showed that telomerase provided a potential target to prevent and treat Giardia Canis disease. Other protozoon disease can also be prevented by adjusting the activity of telomerase.Construction of GCV universial transfer vector. According to transcriptional start site,replication origin and packaging site of Giardia canis virus (GCV) genome (DQ238861),in this study, enhance green fluorescent protein (EGFP) was stably expressed in G. canis mediated by GCV. The plasmid pNEO/GDH/MCS/EGFP, containing the neomycin phosphotransferase (NEO) encoding region flanked by the 636 nt of 5′-terminus and the 2,174 nt of 3′-terminus from GCV positive strand RNA, was constructed by inserting EGFP gene into downstream from the NEO gene and glutamate dehydrogenase (GDH) 5′-terminus uncoding regions on a single plasmid, and its in vitro transcript was introduced into GCV-infected G. canis by electroporation. The transfectants expressed EGFP persistently under G418 selection. This stable transfection system shouLd provide a valuable tool for genetic study of G. canis.Identification of telomeric repetitive DNA sequences of G. canis. According to the resuLt of reported that 5′-TAGGG-3′was the telomeric repetitive DNA Sequences of G. canis, the oligonucleotide probe was designed. Then the genomic DNA of G. canis was hybridizated with oligonucleotide probe. The resuLt indicated that the genomic DNA of G. canis could hybridizate with oligonucleotide probe and zone of hybridization could be seen clearly. With the time of Bal 31 digestion delay, the zone of hybridization on the nylon membrane disappeared. The result showed the telomeric repetitive DNA sequences of G. canis was 5′-TAGGG-3′.Detection of G. canis telomerase activity. According to the modified TRAP, forward primer PF and five reverse primers PR were designed, which because we don't know which is the last basic group to add. At the same time, two negative controls were established during TRAP. The first negative control was cell extracts pretreatmented by 50μg /mL of RNase A for 30 min at 37℃, the second one is the extracts replaced by CHAPS solution, it ensured that the TRAP products could represent the activity of telomerase. The result indicated that reverse primer PR3 couLd be more efficiently combined to the extend products of telomerase. Telomerase activity was detected and we confirmed the high telomerase activity presented in trophozoites not in cysts.The cleavage activity of GCV transfer vector-mediated miniature ribozyme for GcTERT in vitro transcript. In the present study,cDNA encoding miniature ribozyme flanked with various lengths of antisense RNA were cloned into a viral vector pNEO/GDH/MCS. The cleavage activities on GcTERT mRNA in vitro of the two ribozyme TRzS flanked with 21 nt and 20nt GcTERT antisense RNA on each arm and TRzL flanked with 324 nt and 380 nt GcTERT antisense RNA were 76.14% and 83.42% respectively by absolute real﹣time quantitative RT﹣PCR detection. One control group,PTR without the inserted ribozyme showed 31.56%. The anther control group,GRzL flanked with 841 nt and 850 nt GDH antisense RNA showed no effect on GcTERT mRNA in vitro.Inhibition of telomerase activity in Giardia canis by GCV vector-mediated miniature ribozyme. The two ribozyme TRzS and TRzL were introduced into GCV﹣infected G. canis trophozoites by electroporation. GcTERT mRNA in the transfected cells were decreased by 72% with the ribozyme TRzS and 84 %with the ribozyme TRzL,which were detected by relative real﹣time quantitative RT﹣PCR. The two miniature ribozyme transfected cells grew slowly and its telomerase activity is inhibitied greatly. In addition, this study demonstrated the feasibility of using a viral vector to express a ribozyme targeted at a specific mRNA in Giardia to reduce the expression of a specific gene and that GCV transfer vector is an effective tool for the study on gene manupuLation of Giardia.