Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major

• Autor(a) principal: Freitas, Rodrigo de Barros
• Data de Publicação: 2010
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2423
• topic: Hibridomas; E-NTPDase-2; Leishmania major; Hybridomas; E-NTPDase-2; Leishmania major; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

Leishmaniasis are parasitic diseases caused by protozoa of the Kinetoplastida order, Trypanossomatidae family and Leishmania genus. It currently represents a public health problem in Brazil and other countries in the world. The E-NTPDase or apyrases present in such parasites are mainly responsible for the degradation of nucleotides, especially extracellular di- and/or triphosphate, thus allowing the parasite to modulate an immune response in the host, which needs a purinergic signalling, and promoting the cellular development for its participation in the purine salvage pathway, in which the parasites are dependent. Monoclonal antibodies are originated of a hybridoma for the recognition of a specific antigen and can be selected for a desired specificity as well as produced in large scale and with good quality. This research aimed the expression of the heterologous L.major E-NTPDase-2 proteins and the production of monoclonal antibody-secreting hybridomas against such protein. A design of specific primers aiming the amplification of the coding region of the soluble portion (ecto-domain) of the E-NTPDase-2 was proposed, without the coding region for the amino-terminal peptide portion, resulting in a 1053-base-pair DNA. The amplicon was obtained by PCR amplification, cloned into an amplification (pJET-bunt) and expression (pET-21b) vector of bacterial system (E. coli DH5α). The protein expression of the ENTPDase- 2 was carried out in a bacterial system (E. coli BL-21) and the purification was performed by affinity chromatography on a Ni2+-agarose column (Ni-NTA). With the purified protein, immunizations of the mice were conducted with subsequent confirmation of the production of anti-NTPDase-2 antibodies, by means of DOT ELISA. The fusion of the B cells from the immunized mice, with the myeloma cells (Sp20), was performed in order to obtain the hybridoma. Then, hybridoma of L.major anti-E-NTPDase-2 were isolated making its usage possible in several future applications such as the diagnosis and treatment of canine leishmaniasis.