Resumen

MORENO, Luz Yurany; GUERRERO, Carlos Arturo  y  ACOSTA, Orlando. Expression and purification of rotavirus structural proteins VP5* and VP8* in bacteria E. coli BL21(DE3). Rev. colomb. biotecnol [online]. 2013, vol.15, n.1, pp.82-97. ISSN 0123-3475.

The characterization of rotavirus structural proteins and the cell surface proteins involved in virion binding and penetration depends on the availability of substantial amounts of highly purified proteins. The aim of the present work was to express and purify the rotavirus structural proteins VP5* and VP8* in order to produce polyclonal antibodies against them. Recombinant proteins VP5* (rVP5*) and VP8* (rVP8*) were expressed in E. coli cells transfected with pGEX-4T or pET 28a containing their corresponding encoding sequences. Culture medium, bacterial concentration before induction, inductor concentration and induction time were used as variables. The greater proportion of rVP8* was obtained when transfected bacteria were grown in medium LB and induction was started by adding 1 mM IPTG to cells at OD _{600 nm} 0.5 followed by 6 h-induction. The highest proportion of rVP5* was reached when cells at OD _{600 nm} 0.2 were induced with 0.5 mM IPTG for 4 h in medium 2XYT containing 2% glucose. The recombinant proteins accumulated in the insoluble fraction were solubilized with ionic and non-ionic detergents, and purified by affinity chromatography before being used as antigens for production of rabbit polyclonal antibodies. The antibodies produced were characterized through their ability to recognize the corresponding antigens in ELISA, Western blotting, and immunochemistry assays in rotavirus infected cells. The amount and purity of recombinant proteins obtained in this work, and the antibodies against them, are expected to be useful for the characterization of the virus-cell interaction.

Palabras clave : rotavirus; VP5*; VP8*; recombinant proteins; E. coli BL21(DE3).

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