Construction Of TV Transfer Vector And EGFP Expressed Stably In Trichomonas Vaginalis

Trichomonas vaginalis, which parasitizes in urogenital system, is one kind of flagellates causing vaginal inflammation. Trichomonas vaginalis virus(TVV) is a kind of dsRNA virus parasitized in Trichomonas vaginalis. The prior research shows that the existence of TVV has relationship with the invasiveness and Metronidazole resistance of Trichomonas vaginalis. the discovery of TVV shows a new way for T.vaginalis study in molecular biology.In this study, TVV stable transfer vector was constructed and enhance green fluorescent protein was successfully expressed in the Trichomonas vaginalis. According to feature of T. vaginalis virus (TVV) genome in China and on the basis of TVV transient transfer vector we have constructed, a system named pTVV289/NEO+MCS/TVV174 was developed here in this organism by flanking the NEO gene with the fragments of TVV positive-strand RNA. pTVV289/NEO+EGFP/TVV174 was constructed by inserting EGFP gene into downstream from the NEO gene through MCS, and its in vitro transcript was introduced into T.vaginalis by electroporation. The transfectants expressed EGFP persistently under G418 selection. The results indicated that chimera pTVV289/NEO+EGFP/TVV174 appeared fluorescent signal under fluorescence microscope at 488nm, and without attenuating after the 24th passage .This stable transfection system should provide a valuable tool for genetic study of T.vaginalis.In this study, we also construced DNA stable transfer vector. At first job was to clone and evaluate transcriptional activity of a-succinyl CoA synthetase B (a-SCSB) gene promoter from Giardia lamblia. The promoterα-SCSB5 and poly(A) signalα-SCSB3 ofα-SCSB gene from Trichomonas vaginalis were amplified by PCR and inserted into the pMD18-T vector, resulting in recombinant plasmid pα-SCSB 5 and pα-SCSB 3. The promoterα-SCSB 5 and poly(A) signalα-SCSB 3 were used to constructed EGFP expression cassette pα-SCSB5/EGFP/Eα-SCSB3. The plasmid was electroporated into Trichomonas vaginalis trophozoite and the transfectants appeared fluorescent signal under fluorescence microscope at 16 hours post-transfection, indicating that the promoter we had cloned had transcriptional activity. Then, we established and identificated G418 resistant Trichomonas vaginalis strain. NEO resistant cassette pα-SCSB 5/NEO/Nα-SCSB 3 was construct in which its upstream was the promoterα-SCSB 5 ofα-SCSB gene from Trichomonas vaginalis and its downstream was poly(A) signalα-SCSB3. The minimal lethal dose (MLF) of Trichomonas vaginalis to G418 was determined to be 450μg/mL. Linearized pα-SCSB 5/NEO/Nα-SCSB3 was electroporated into Trichomonas vaginalis trophozoite. The transfected cells would be survived in further culture medium containing G418 as the NEO gene could express G418 resistant products and the G418 resistant cell line was established successfully under G418 selection. The identification of the G418 resistant cell line was conducted by polymerase chain reaction with the specific primers of NEO gene, indicating that linear transfected DNA was integrated into the cell genome. Establishment G418 resistant cell line should greatly enhance our ability to study stable DNA transfection of Trichomonas vaginalis. Last, we established a stable DNA transfection of Trichomonas vaginalis. Recombinant plasmid pNEO/EGFP was constructed by combining the EGFP expression cassette and the NEO resistant cassette, this plasmid was linearized and electroporated into Trichomonas vaginalis, and the transfectants were selected by G418. The G418 resistant cell line was established successfully. The NEO and EGFP gene were detected by PCR, demonstrating that they were integrated into Trichomonas vaginalis genome and their transcriptions were all synthesized persistently under the instruction ofα-SCSB promoter. Fluorescent signal detected under fluorescence microscope at 488nm indicated that fluorescent signal without attenuating after the 17th passage. The stable DNA transfection system have been constructed and it should provide a valuable tool for TVV vector construction.