Purification of Immunogenically Active Ribonucleic Acid Preparations of Salmonella typhimurium : Molecular-Sieve and Anion-Exchange Chromatography

Abstract

Immunogenic Salmonella typhimurium ribonucleic acid (RNA) preparations, prepared by differential centrifugation, phenol extraction at 65 C, and ethanol precipitation from 0.5% sodium dodecyl sulfate solution, maintained their immunogenicity through lyophilization. As measured by survival, differential pathogen counts 5 days postchallenge, or clearance of the infecting organism from the tissues, immunization with 50 μg (dry weight) of the lyophilized preparation proved as effective as immunization with 0.1 LD 50 of attenuated S. typhimurium cells. Chromatography of the immunogenic fraction through Biogel P-6 (exclusion limit > 4,600) or through Biogel P-300 (exclusion limit > 300,000) resulted in only one immunogenically active protein of the eluate found in the void volume of the columns. Diethylaminoethyl (DEAE) cellulose anion-exchange chromatography of the RNA preparations showed that the immunogenic activity was eluted from the column at 0.8 to 1.0 m NaCl in a linear 0.1 to 2.0 m NaCl gradient. Nonimmunogenic, protein-containing minor peaks were eluted at 0.1 to 0.5 m NaCl. Serial fractionation of the crude RNA preparations over Biogel P-6 to DEAE cellulose to Biogel P-300 molecular-sieve or anion-exchange columns did not alter the immunogenicity of the RNA preparation. Incorporation of the column fractions into Freund's incomplete adjuvant did not increase their relative effectiveness in eliciting anti-salmonella resistance. Chemical analysis of the immunogenic preparations indicated that they were lacking in detectable protein, lipid, and deoxyribonucleic acid. These results suggest that the immunogenic moiety of the crude nucleic acid fraction is either RNA or an as yet undefined polysaccharide of greater than 300,000 molecular weight.