Molecular Cloning, Expression and Characterization of Para Flagellar Rod Protein 1 of Trypanosoma evansi
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.
2. Singh V, Singh A, Chhabra MB. Polypep-tide profiles and antigenic characterization of cell membrane and flagellar prepara-tions of different stocks of Trypanosoma evansi. Vet Parasitol. 1995; 56(4):269-79.
3. Donelson JE, Kent HK, EL-sayed MN. Multiple mechanisms of immune evasion by African trypanosomes. Mol Biochem Parasitol. 1998; 91: 51-56.
4. Maharana BR, Tewari AK, Singh V. An overview on kinetoplastid paraflagellar rod. J Parasit Dis. 2015; 39(4):589-95.
5. Portman N, Gull K. The Paraflagellar rod of Kinetoplastid Parasites: From Structures to components and function. Int J Parasi-tol. 2010; 40(2):135-48.
6. Maga JA, Sherwin T, Francis S et al. Ge-netic dissection of the Leishmania parafla-gellar rod, a unique flagellar cytoskeleton structure. J Cell Sci. 1999; 112 ( Pt 16):2753-63.
7. Lanham SH, Godfrey DG. Isolation of salivarian trypanosomes from man and other animals using DEAE-cellulose. Exp Parasitol. 1970; 28(3):521-34.
8. Sambrook J, Russel DW. Molecular clon-ing: A laboratory manual 3rd ed. Cold spring Harbor Laboratory Press, Cold spring Harbor, NY. 2001.
9. Laemmli UK. Cleavage of structural pro-teins during assembly of the head of bac-teriophage T4. Nature. 1970; 227(5259):680-5.
10. Hall T. BioEdit: important software for molecular biology. GERF Bul Bio. 2011; 2:60-61.
11. Saitou N, Nei M. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Bio Evol. 1987; 4(4):406-25.
12. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution. Mol Biol Evol. 2016; 33(7):1870-4.
13. Gasteiger E, Hoogland C, Gattiker A et al. Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walk-er (ed): The Proteomics Protocols Hand-book, Humana Press. 2005; 571-607.
14. Yang C, Suo X, Huang X et al. Protection of mice against homologous or heterolo-gous infections with antiserum mixture to the predominant variable antigen type rep-ertoire of Trypanosoma evansi YNB stock. Exp Parasitol. 2007; 116(1):53-8.
15. Maharana BR, Rao JR, Tewari AK et al. Molecular characterization of Para Flagellar Rod Protein Gene (PFR) of in Trypanosoma evansi. J Appl Anim Res. 2014; 1: 1-5.
16. Abdille MH, Li SY, Jia Y et al. Evidence for the existence of paraflagellar rod pro-tein 2 gene in Trypanosoma evansi and its conservation among other kinetoplastid parasites. Exp Parasitol. 2008; 118(4):614-8.
17. Luhrs KA, Fouts DL, Manning JE. Im-munization with recombinant paraflagellar rod protein induces protective immunity against Trypanosoma cruzi infection. Vaccine. 2003; 21(21-22):3058-69.
18. Saravia NG, Hazbón MH, Osorio Y et al. Protective immunogenicity of the Parafla-gellar rod protein 2 of Leishmania mexicana. Vaccine. 2005; 23(8):984-95.
19. Wrightsman RA, Miller MJ, Saborio JL, Manning JE. Pure paraflagellar rod protein protects mice against T.cruzi infection. In-fect Immun. 1995; 63(1):122-5.
20. Desquesnes M, Bosseno MF, Brenière SF. Detection of chagas infections using Tryp-anosoma brucei crude antigen demonstrates high cross-reactions with Trypanosoma cruzi. Infect Genet Evol. 2007; 7(4):457-62.
21. Clark AK, Kovtunovych G, Kandlikar S et al. Cloning and expression analysis of two novel paraflagellar rod domain genes in Trypanosoma cruzi. Parasitol Res. 2005; 96(5):312-20.
|Issue||Vol 13 No 4 (2018)|
|Para flagellar rod pro-tein 1 SDS-PAGE Trypanosoma evansi Western blot|
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