Produção e purificação de toxinas e antitoxinas clostridiais

Abstract

The objective of this work was to produce, purify and analyze the toxins and antitoxins C and D from Clostridium botulinum, beta and epsilon from Clostridium perfringens, as well as to compare them to the existing standards used by the official organs, aiming their uses in research for the production of new vaccines, development of alternative diagnostic methods and quality control of vaccines. The production of the toxins was carried out by fermentation of the strains in specific media for each species in a bench bioreactor. C. botulinum C and D toxins were purified by gel filtration. C. perfringens type C beta toxin was purified by immobilized-metal affinity chromatography using zinc as a chelating agent. C. perfringens type D epsilon toxin was purified by anion exchange chromatography. Immunoglobulins G and Y were produced by multiple immunization of sheep and chickens, respectively, with inactivated purified toxins in Freund's adjuvant. Subsequently, IgG were purified by precipitation by caprylic acid and ammonium sulfate, and the IgY were purified by ammonium sulfate precipitation. C. botulinum C and D toxins were purified to the level of several SDS-PAGE gel bands, which represent the toxins, their subunits and accessory proteins associated. C. perfringens type C beta toxin was purified to the level of a high intensity gel band, and few bands of high molecular weight and low intensity which may be oligomerized forms of the toxin. C. perfringens type D epsilon toxin was purified to the level of a very high intensity gel band, and few bands of high molecular weight and very low intensities. The standard toxins produced by this study showed higher yields of protein and higher purity when compared to the official standards. The produced and purified IgG and IgY obtained high titers, revealing the quality of the immunogens and purification methods. In addition, they presented high avidity indexes, higher than the indexes of the official standards, indicating the superior quality of the products generated by this study. The present work produced clostridial toxins and antitoxins with high purity levels and superior quality when compared to the official national ones, and they can be applied for the development of new diagnostic methods, vaccines and methodologies for quality control of vaccines.