The Preparation Of Transcript In Vitro And Transfection To Giardia Canis Free From Giardiavirus

The Giardia canis virus (GCV) is a 6.2 kb double-stranded RNA (ds RNA) virus, which obligatory parasitism in G. canis trophozoites. In this study, three pairs of Specific overlapping primers were designed, and the cDNA of infective GCV was obtained, and then the transcript in vitro was prepared and was used to infect the virus-free isolate of G. canis trophozoites. The RNA transcripts were transfected into the virus-free G. canis trophozoites through electroporation. The other infection is that the virual particles was extracted by Millipore filtration from G. canis growth medium, and then incubated with the virus-free isolate of G. canis trophozoites. The ultramicrostructure of the infected cells was observed every 12h. The virual particles can both be detected by this two ways through extracting total nucleic acid and observating its ultramicrostructure. The research will contribute to study on the relations of the virus and protozoon and host and will establish the foundation to further study of GCV.To get the total cDNA of GCV, three pairs of Specific overlapping primers were designed, with T7 RNA promotor in 5′- F1. Three segments were got by RT-PCR and were linked together by pfu DNA Polymerase, at last the total cDNA of GCV was obtained. Its homology was analyzed with GVC cDNA sequence reported in GenBank, the result proved that there is a significant homology. The two flush ends were added by"A"and it become adhesive ends, then they were linked to pGEM-T vector. The GVC cDNA was constrcted and provid templets for the preparation of transcript in vitro.To preparation of transcript in vitro and transfection to viral-free G. canis trophozoites, the plasmid pGCV was linearized at the 3′end with XhoI and the transcription reactions with T7 RiboMAXTM Express Large Scale RNA Production System were performed. The transcript of pGCV was electroporated into the GCV-free strain of Giardia trophozoites. The ultramicrostructure was observated, and the total nucleic acid of infected cells was extracted after 72h, Samples were analyzed by 0.8% agarose gel, the virual particles and the objective strap can be detected respectively. GCV's successful transfection will provid useful evidence for study on the character of GCV.To observate its ultrastructure of G. canis trophozoite, the virus-free isolate of G. canis trophozoites was infected by two ways. the RNA transcripts were transfected into the virus-free G. canis trophozoites through electroporation is the one way, the other way is that the virual particles was extracted by Millipore filtration from G. canis growth medium, and then incubated with the virus-free isolate of G. canis trophozoites. the cell growth state was Observed at 12h, 24h, 36h, 48h, 60h and 72h after infection respectively , compared with the state that in normal condition and cell growth curve was drawed. Meanwhile, the ultramicrostructure observed at 12h, 24h, 36h, 48h, 60h and 72h after infection. As a result, the virual particles in the cells that infected by two ways can be detected after 36h;The infected cells grew slowly and their internal structures got blurred in early phase of infection;there were many viral particles which is like grape in the cytoplasm after 48h;most of the viral particles were detected in cytoplasm at 36h and after 72h the viral particles also appeared in the two nuclei. the infection of G. canis by the virus caused severe cellular vacuolization;there is no obviously difference in cellular structure between the infected cells and viral-phore cells. It will provide the foundations for the further study on the routes of infection,replication, expression and the opathogenic mechanism of viruses,and will provid directions to the study on the relations of the virus and protozoon and host.