Construction Of TVV Transfer Vector And EGFP Expressed In Trichomonas Vaginalis

Trichomonas vaginalis, which parasitizes in urogenital system, is one kind of flagellates causing vaginal inflammation. The patients run a six-fold higher risk of HIV infection. Trichomonas vaginalis virus(TVV),is a kind of ds RNA 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 Trichomonas vaginalis virus(TVV) shows a new way for T.vaginalis study in molecular biology. At present, the vector system reconstructed from GLV has been used in several area ,for example : expression of exogenous gene, structure and function analysis of G.. lamblia gene. It has been a significant tool for the study of G.. lamblia in molecular biology.While the study about TVV vector is relatively lagged. Chimera of TVV cDNA and enhanced green fluorescent protein (EGFP) were constructed and their in vitro transcript were electroporated into TVV-infected T. vaginalis trophozoites, the control signals of the TVV genome required for expression of foreign gene were preliminary determined according to the expression level of EGFP in the transfected parasites. This transfection system should provide a valuable tool for genetic study of T. vaginalis.Construction of GCV Transfer Vector According to feature of T. vaginalis virus (TVV) genome in China,a system was developed here for expression of a foreign gene in this organism by flanking the enhanced green fluorescent protein(EGFP) gene with the fragments of TVV positive-strand RNA . Chimera pTVVL289/EGFP,pTVVL289/EGFP/TVVS,pTVVL559/EGFP/TVVS were constructed.Expression of EGFP mediated by TVV Transcript of the Chimera pTVVL289/EGFP,pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS were synthesized in vitro with T7 RNA polymerase and used to transfect T. vaginalistrophozoites already infected with TVV. The results indicated that chimera pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS appeared fluorescent signal under fluorescence microscope at 488nm, and without attenuating after the fifth passage .We detected the EGFP mRNA by RT-PCR in transfected T. vaginalis and the results of SDS-PAGE demonstrated that EGFP was expressed in T. vaginalis and mainly secreted to medium supernatant. while chimera pTVVL289/EGFP did not appear fluorescent signal under fluorescence microscope, it shows that the 3'UTRs of TVV play a part in replication,translation and/or package of virus particle.