Antibody delivery into viable epimastigotes of Trypanosoma cruzi as a tool to study the parasite biologyReportar como inadecuado




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American trypanosomiasis is a zoonosis of worldwide medical importance and currently there is no effective treatment in chronic patients, hence the importance of the study of protein function of the parasite with the objective of finding new drug targets and to know better the biology of the agent causal Trypano-soma cruzi. T. cruzi is an RNAi-negative parasite, therefore the silencing genes strategies by RNAi is not possible; for that reason, antibodies may be taken as a tool for studying the parasite proteins function by blocking these molecules with specific antibodies. The aim of this work was to establish a methodology for antibody delivery antibody transfection into viable parasites. We used anti-cyclin-A antibody human origin in western blot assay with epimastigote of T. cruzi proteins and this recognized a ~55 kDa polypeptide. Several methods for antibody transfection electroporation, saponin permeabilization and a lipid-based formulation were tested. The first two methods were unsuccessful. In electroporation was impossible to visualize the antibody inside parasites and with saponin permeabilization, antibodies were successfully introduced, but with loss of parasites viability. The lipid-based formulation method forms noncovalent complexes with antibodies. These complexes are internalized by cells and antibodies are released into the cytoplasm. With this method, a successful antibody delivery was achieved. Anti-cyclin antibodies were visualized in the cytoplasm from fixed transfected parasites immunofluorescence assays. At 24 h post-transfection, parasites maintained their viability 90% and were able to arrest the cell cycle in G0-G1-phase of cultured epimastigotes cell population increased in G0-G1-phase from 50.5% to 66.2% and decreased in S-phase from 47.2% to 26%. It was also observed that anti-cyclin-A antibodies inhibit the parasite population doubling p < 0.05, 95% CI. This is the first report of antibody-delivery into viable epimastigote forms of T. cruzi, with a simple and cheap technique, which will allows carrying out further studies of this protozoan.

KEYWORDS

Trypanosoma cruzi; Antibody Delivery; Antibody Transfection; Cell Cycle; Anti-Cyclin-A Antibody

Cite this paper

Acosta-Viana, K. , Julio, H. , Matilde, J. , Eugenia, G. and Rosales-Encina, J. 2013 Antibody delivery into viable epimastigotes of Trypanosoma cruzi as a tool to study the parasite biology. Advances in Bioscience and Biotechnology, 4, 719-726. doi: 10.4236-abb.2013.46095.





Autor: Karla Y. Acosta-Viana, Huchin-Cetz Julio, Jimenez-Coello Matilde, Guzman-Marin Eugenia, Jose L. Rosales-Encina

Fuente: http://www.scirp.org/



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