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Caporale GMM. Purification of subunits of rabies virus by chromatography [ Ph.D. in Biotechnology]. São Paulo (Brazil): Instituto de Ciências Biomédicas da Universidade de São Paulo; 2010. The subunits of the rabies virus can be purified by different methodologies and may have several applications for both laboratorial diagnosis and research of rabies disease. The method of ultracentrifugation in cesium chloride (CsCl) gradient enables the achievement of concentrations semi purified ribonucleoprotein (RNP) of rabies virus. Another widely used technique for protein purification is chromatography, which has been used in the purification of rabies virus grown in cells, for vaccine production. The aim of this study was to establish methodology for purification of RNP by immunoaffinity chromatography, to increase the purity of the protein and reduce the operating time of the process. After selection among viral strains, PV (Pasteur Virus) and CVS (Challenge Virus Standard) held in BHK-21 cells (Baby Hamster Kidney), were prepared batches of these cells infected with CVS strain selected for having presented fluorescence intensity than PV strain, maintained for 48 hours in static or roller system for extraction of RNP from the cell lysis. The concentration and semi purification of RNP were performed by ultracentrifugation in CsCl gradient, to immunize rabbits and obtain hyperimmune sera, of which the antibodies were purified by ion exchange chromatography and affinity (protein A). Immunoglobulins (Ig) were purified then coupled to Sepharose 4B activated with cyanogen bromide, which was packaged and used for purification of the RNP, by passing the cell lysate samples on immunoaffinity column, in cycles in a flow of 1 mL/min in Äkta Purifier system. After cells lysis of grown in static system and roller were obtained 15 mL and 100 mL of protein solution, respectively, and both RNP solutions, after ultracentrifugation, showed satisfactory concentrations of protein for animal immunization. The hyperimmune serum with highest titer of antibodies anti RNP (1:2600), when purified by affinity chromatography, allowed the obtainment of IgG with high purity and protein concentration of 3.6 mg/mL, 19.0 mL. The efficacy was of 78% in the coupling reaction of antibodies to Sepharose 4B gel, allowing the packing column of 0.9 mL. Samples of cell lysate containing RNP, subjected to purification on immunoaffinity column, resulted in fractions of RNP with protein concentration of 0.096 mg/mL for the first cycle and 0.275 mg/mL for the second cycle and in electrophoresis was observed the band corresponding to molecular mass of nucleoprotein (57 kDa). The methodology of immunoaffinity chromatography has been successfully established and provided obtaining of RNP with high purity and reduction of operating time.

Purificação de subunidades do vírus da raiva por meio de cromatografia.