Effect On Rab11c Gene Expression In Trichomonas Vaginalis By TVV Transfer Vector-mediated Hammerhead Ribozyme

Trichomonas vaginalis is a flagellated, parasitic protozoan which causestrichomoniasis or urethritis by infecting urogenital tract. T. vaginalis was discoveredby Alfred Donne in1836. Trichomoniasis is one of the most common causes ofnonviral genitourinary sexually transmitted infection (STI) in human with aworldwide prevalence of170million cases annually. Despite the high prevalence, it isone of the poorly studied parasites with respect to virulence properties, pathogenesisand immunopathogenesis. Trichomonas vaginalis virus(TVV) is a kind of dsRNAvirus parasitized in T.vaginalis. The prior research shows in T.vaginalis that the TVVhas effected on pathogenicity,toxicity,drug resistance and so on.The TVV based RNA transfection systerm established had provide a new wayfor study of genetie and protein in T.vaginalis. Since our group through iTRAQtechnique obtain the differential protein between TVV-infected and TVV-freeT.vaginalis. Rab11C has been screened out. The Rab family is part of the Rassuperfamily of small GTPases. Rab GTPases are key regulators of G-proteins which isinvolved in the function of morphological transformation in pathogenesis of T.vaginalis.Construction of TVV transfer vector-mediated hammerhead ribozyme forRab11C and the cleavage activity of it in vitro transcript. In this study,we useliver-serum medium to cultivate TVV-infected and TVV-free T.vaginalis in vitro. Weconstructed a special hammerhead ribozyme to cleavage Rab11C,which connectedwith the TVV based RNA transfection systerm giving rise to the Plasmid pNME/RLand pNME/RS. The cleavage activity of in vitro transcribed hammerhead ribozymeagainst Rab11C was analyzed by real-time quantitative RT-PCR, the cleavage activityon Rab11C mRNA was82.12%and80.2%respectively, and the plasmid pNME/RAwithout the inserted ribozyme cleavage activity on Rab11C mRNA was just32.69%,which provided a theoretical basis for the test in vivo.Inhibition of Rab11C activity in T.vaginalis by the ribozyme. The tworibozyme pNME/RL and pNME/RS were electroporated into TVV-infectedT.vaginalis trophozoites. Rab11C mRNA in the transfected cells were decreased by73%with the ribozyme pNME/RS and82%with the ribozyme pNME/RL,whichwere detected by relative real-time quantitative RT-PCR. Detection the effects on thetranfected T.vaginalis,which through observed them under microscope and calculatethe number. The tranfected T.vaginalis grew slowly, morphous transformed andhypergasia,indicate the Rab11C has particular influence on parasite growth andmorphous. In the meantime,detection the expression of TVV by relative real-time quantitative RT-PCR, the results shows the expression of TVV had decreased by21days after expression of Rab11C was inhibited.The TVV transfer vector-mediated hammerhead ribozyme for Rab11C inTVV-infected T.vaginalis trophozoites were constructed to validate the expressionlevel of Rab11C gene and its influence on TVV and growth of T.vaginalis.This studyprovided a basis for further study on diagnosis,pevention and control ofTrichomoniasis, molecular biology and other fields of T.vaginalis.