Molecular Cloning, Expression and Characterization of Para Flagellar Rod Protein 1 of Trypanosoma evansi

Abstract

Background: Antigenic variation allows the trypanosomes to evade the potentially destructive host immune response and is an important reason for failure to develop a protective vaccine. Among the non-variant structural proteins, paraflagellar rod protein (PFR) is a prospective vaccine target owing to its role in the active movement of the parasite. Methods: The PFR1 gene was cloned in pET-32a expression vector and after confirmation by restriction digestion, expressed as a Histidine-tagged fusion protein, in BL21 DE3 strain of E. coli. The expressed protein was affinity purified and then renatured. The immunoreactivity of the expressed recombinant protein was shown by western blot analysis using the specific serum. The experiment was carried out during 2013-14 at Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, U.P., India. Results: The results of sequencing, restriction digestion analysis, and PCR reaction revealed that cloning of PFR1 gene in pET-32a expression vector and the results of SDS PAGE and Western blot further confirmed its homogeneity and purity. The in silico Te-PFR1 (T. evansi PFR1) nucleotides sequence analysis revealed its close homology with the other members of the order Kinetoplastida. Conclusion: We report here the molecular cloning, heterologous expression, and characterization of PFR1, a constituent protein of PFR. Due to its conserved nature, the PFR1 protein could be a prospective vaccine target against multiple Trypanosoma species.

Keywords

• Para flagellar rod pro-tein 1
• SDS-PAGE
• Trypanosoma evansi
• Western blot