Comparison Of Differential Protein Between TVV Infection And TVV Free Trichomonas Vaginalis

Trichomonas vaginalis (T. vaginalis) causes urogenital system infections in humans all over the world.The symptom of T. vaginalis infection included vaginal inflammatio in females and urinary tract or reproductive system in males. It could also cause other animals such as mice and squirrel monkeys.It was a danger factor to the health of people and animal,which was an important parasitic protozoa. Trichomonas vaginalis virus(TVV) is a kind of dsRNA virus,which parasitized in T.vaginalis. The TVV discovered shows a useful way for T.vaginalis in molecular biology study. The TVV based RNA transfection systerm established should provide a useful tool for genetic and protein study of T.vaginalis.Although some reseaches has indicated that the TVV infection has effected on pahtogenicity,toxicity,drug resistance and so on in T.vaginalis,there was no study on the differential expression of overall protein between TVV-infected and TVV-free T.vaginalis.In a word, the present study,it was the first research to compare the defferential expression of protein between the TVV-infected and TVV-free T.vaginalis.In this study,we used ox liver medium to cultivate TVV-infected and TVV-free T.vaginalis with in vitro. Meanwhile, virus particle was purified with CsCl to eliminate the influence of TVV on the examination. The overall protein were extracted from TVV,TVV-infected and TVV-free T.vaginalis by ultrasonication,respectively; Through iTRAQ technique, nanoLC separation and data analysis to obtain the differential protein between TVV-infected and TVV-free T.vaginalis;Then, we utilized real time PCR method to detect the differential level of TVTPI mRNA and electroporate method to introduce hammerhead ribozyme into T.vaginalis to validate the function of TVTPI.A total of 270 proteins were screened out,included 50 differential expression proteins,which included some kinds of protein.For the most part of proteins were up regulated in TVV infected T.vaginalis; With real-time PCR detecting,the level of TVTPI mRNA was 58.00% in TVV-free than TVV-infected T.vaginalis; At the same time, we constructed a special hammerhead ribozyme to cleavage for TVTPI,connected with the TVV based RNA transfection systerm giving rise to the plasmid pNEO/73nt. In vitro,the cleavage activity on TVTPI mRNA was 82.19%, and the plasmid pNEO/TPI cleavage activity on TVTPI mRNA affected small just was 35.5%,which provided a theoretical basis for test in vivo.The ribozyme pNEO/73nt was introduced into TVV infected T.vaginalis by electoporation.TVTPI mRNA level decreased 75.20%, which detected by real time quantitative PCR.The tranfected T.vaginalis grew slowly and shape transformed. So the TVTPI has played significant effect on paraite growth.The test provided a basis for further study on pathogenic mechanisms, drug resistance,virulence and protemomics research of T.vaginalis.