Resumo

MAYORGA, Diana; RUBIO, Linda; GUERRERO-FONSECA, Carlos A  e  ACOSTA-LOSADA, Orlando. β3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro. rev.fac.med. [online]. 2010, vol.58, n.2, pp.115-127. ISSN 0120-0011.

Background. Commercial integrin β3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin β3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins β3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins β3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins β3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electroelution from a gel slice. Polyclonal antibodies against protein β3 were generated in rabbit. Incubation of eluted proteins β3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins β3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against β3 in addition to the establishment of the in vitro interaction of proteins β3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small intestinal villi from suckling mice.

Palavras-chave : proteins; blood platelets; rotavirus; electrophoresis.

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