Cloning and biological activity determination of sialidase gene of trichomonas vaginalis

Objective: The aims of the research were cloning of sialidase gene from T.vaginalis which known as a parasitic protozoon and investigation of biological activity of both crude and purified enzyme preparations obtained from recombinant organism. Material and Methods: In this research, T. vaginalis genomik DNA sample was provided by ADÜ Medical Parasitology Department. PCR amplified sialidase gene transferred to E.coli host after restriction and ligation steps. Then, sialidase activity was tested on both crude and purified samples using Neurominidase assay kit. Results: PCR amplified sialidase gene was 1050 bp in length. According to sequence analysis, amplicon sequence showed highest identity to T.vaginalis G3 sialidase gene at 99.69%. The clone named Sia32 was choosen after transformation steps and molecular weight of recombinant sialidase protein was determined as 44.5 kDa. According to optimization results, most effective way for protein purification was the one which started without imidazole and elution at 25-200 mM imidasole. To determine the biological activity of enzyme in crude and pure samples, neurominidase assay kit was used. It was found that enzyme activity was lower in purified samples than the crude ones. Conclusion: In this study, T.vaginalis sialidase gene was cloned and the biochemical activity of enzyme was verified with commercial assay kit. Thus, a recombinant sialidase producing E.coli BL21 strain was constructed in our laboratory. Recombinant strain will be a source for further studies regarding other biological properties of enzyme and inhibitory substance development for sialidase.