Inhibition Of KRR1 Gene Expression In Giardia Canis By GCV Transfer Vector-mediated Hammerhead Ribozyme

Giardia canis,an early eukaryote that infects several species including humansand a major agent of waterborne diarrhea throughout the world,can be infected with adouble﹣stranded RNA virus,Giardia canis virus (GCV). In this study,Giardia canistrophozoites infected with GCV in vitro cultivation was successfully established;andthe full﹣length cDNA sequence of Giardia canis virus was analyzed, GiardiaCanis virus transfer vector was constructed and green fluorescent protein wassuccessfully expressed in the Giardia canis,two hammerhead ribozyme mosaicismGiardia canis virus transfer vector KRzS or KRzL were firstly constructed and thecleavage activity of Giardia canis virus﹣mediated hammerhead ribozyme for KRR1in vitro transcript and the effect on inhibition of KRR1 gene expression in Giardiacanis by GCV transfer vector -mediated hammerhead ribozyme were studied.Establishment of in vitro Cultivation of Giardia Canis Infected with GCV The5-day-old suckling gerbil was infected with the cysts of Giardia canis which wereisolated from dogs in changchun and purified by sucrose density gradientcentrifugation-G1 acid funnel filtration method. Trophozoites were isolated asepticallyfrom the duodenum of the infected animals after 8 days,then transferred to modifiedTYI-S-33 medium and cultivated at 37℃.The trophozoites were centrifuged with5000×g for 15min after LIN freeze﹣thawing three times and the supernatant stainednegatively by phosphotungstic acid was observed with transmission electronmicroscope. The results indicated that the Giardia canis trophozoites adapted graduallyto environment and grew a cellular monolayer after 14 days and were detected byfreezing and thawing experiment,purity quotient,stability,biology characteristics andmicrobial contamination detection.The results demonstrated that a stable cell strainhad been established. Giardia canis virus with icosahedron spherical shape and 36nmin diameter was observed by electronmicroscope. In vitro cultivation of Giardia canistrophozoites with GCV is successfully established,which is crucial to understandingthe molecular biology of GCV.The full﹣length cDNA Sequence Analysis of Giardia Canis Virus According tothe published nucleotide sequence of giardiavirus,six pairs of primers were designedand total nucleic acids from trophozoites of Giardia.canis isolated from dogs inChangchun were used as template for RT﹣PCR. After RT﹣PCR, the products werelinked into pMD18﹣T vector,then cloned,sequenced and analyzed. The Genomesequence of Giardia canis virus was 6 276bp(DQ238861),which revealed the presenceof two large open reading frames that are separated by a ﹣1 frameshift and share anoverlap of 220 nt. The ORF1(367﹣3030) encodes a polypeptide of 887 amino acidresidues. The ORF2(2808﹣5981)encodes a polypeptide of 1 056 amino acid residues,which contains all consensus RNA﹣dependent RNA polymerase sequence motifs. Theratio of G+C in the Genome was 49.62%. The homology of Giardia canis virus to thatof sequences( L13218) was 94.62% in the nucleotide, and 93.50% in the amino acidlevels.Compared with the sequences (AF525216)was 98.88% in the nucleotide,and98.30% in the amino acid levels.Construction of GCV Transfer Vector and GFP Expressed in Giardia CanisAccording to transcriptional start site,replication origin and packaging site of Giardiacanis virus (GCV) genome (DQ238861),a system was developed here for expressionof a foreign gene in this organism by flanking the green fluorescent protein(GFP) genewith the fragments of GCV positive-strand RNA,transcript of the construct wassynthesized in vitro with T7 polymerase and used to transfect GCV-infectedtrophozoites by electroporation. The results indicated that GFP in electroporated cellspeaked at 20 h after electroporation. The engineered Giardia canis virus transfer vectorcan be successfully used to introduce and efficiently express a heterologous gene inthis eukaryotic microorganism,which demonstrated that the RNA transfer vector mayopen the way for the study on gene expression and regulation of Giardia.The Cleavage Activity of GCV Transfer Vector-mediated Hammerhead Ribozymefor KRR1 in vitro Transcript Giardia,a most primitive eukaryote,has KRR1 proteinresponsible for ribosome biosythesis. In the present study , cDNA encodinghammerhead ribozyme flanked with various lengths of antisense RNA were clonedinto a viral vector pGCV634/GFP/GCV2174 derived from the genome of Giardia canisvirus (GCV). The cleavage activities on KRR1 mRNA in vitro of the two ribozymeKRzS flanked with 21 nt KRR1 antisense RNA on each arm or KRzL flanked with 288nt and 507 nt KRR1 antisense RNA were 73.99% and 81.14% respectively by absolutereal﹣time quantitative RT﹣PCR detection.The two control groups,PKR without theinserted ribozyme or TRzL flanked with 324 nt and 380 nt triosephosphate isomerase(Tim) antisense RNA showed no effect on KRR1 mRNA in vitro.Inhibition of KRR1 Gene Expression in Giardia Canis by GCV TransferVector-mediated Hammerhead Ribozyme The two ribozyme KRzS and KRzL wereintroduced into GCV﹣infected G. canis trophozoites by electroporation. KRR1 mRNAin the transfected cells were decreased by 50%~67% with the ribozyme KRzS and47%~86% with the ribozyme KRzL,which were detected by relative real﹣timequantitative RT﹣PCR. The two hammerhead ribozyme transfected cells grew slowlyand its internal structures got blurred and the cells were deformed, which indicatedthat Giardia may not be able to survive with a total elimination of KRR1 mRNA. Inaddition,this study demonstrated the feasibility of using a viral vector to express aribozyme targeted at a specific mRNA in Giardia to reduce the expression of a specificgene and that GCV transfer vector is an effective tool for the study on cell organicevolution and gene manupulation of Giardia.