ABSTRACT
Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system
Subject(s)
Animals , Female , Mice , Schistosoma mansoni/immunology , Recombinant Proteins , Antibodies, Helminth/physiology , Helminth Proteins/physiology , Plasmids , Recombinant Proteins/isolation & purification , Carrier Proteins , Helminth Proteins/isolation & purification , Blotting, Western , Amino Acid Sequence , Vaccination , DNA, Complementary , Models, Animal , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Fatty AcidsABSTRACT
Commercial preparations of antivenoms and antitoxins produced in horses, that are essentially pure F (ab)2 immunoglobulin preparations, were submitted to ion-exchange chromatography. For anticrotalic, anti-bothropic and anti-tetanic sera it is possible to remove 40-60% of the inactive globulins
Subject(s)
Antitoxins , Antivenins , ChromatographyABSTRACT
A quantidade de pepsina no tratamento do soro com vistas a obtençäo de um bom rendimento da antitoxina é aproximadamente 1000 vezes maior do que a requerida para a remoçäo da fraçäo FC. Comparando-se a atividade de várias preparaçöes de pepsinas comerciais, e fraçöes obtidas por cromatografia DEAE observou-se que o rendimento está relacionado à atividade em pH3.2, o que é devido a parapepsinas. Os resultados sugerem que o soro antitoxina pode ser processado com pepsina purificada, com um bom rendimento e menor contaminaçäo a partir de extrato gástrico de suínos
Subject(s)
Antitoxins , Antivenins , Pepsin A/isolation & purification , Crotalid Venoms , Chromatography, DEAE-CelluloseABSTRACT
A imunofluorescencia indireta (IFI) de sorotipos enteropatogenicos classicos e invasores de E. coli e de Shigella foi comparada com os metodos tradicionais de coprocultura e soroaglutinacao. Os resultados da IFI concordaram com os da coprocultura em 128 dos 140 casos testados para E.coli enteropatogenica (91%) e em 108 dos 112 testados para Shigella (96%). Todos os casos com reacoes positivas por coprocultura foram confirmados por IFI. No grupo controle, onde nao haviam sido isolados tais patogenos por coprocultura, foram evidenciados por IFI, 12 casos com reacoes positivas para E. coli enteropatogenica e 4 para Shigella, incluindo-se 2 com infeccao mista: E. coli 026/Sh. dysenteriae e E. coli 0124/Sh. dysenteriae. Foi discutida a alta sensibilidade e especificidade da IFI quando comparada aos metodos tradicionais, sendo sugerido o valor desta tecnica em estudos epidemiologicos envolvendo os microrganismos em questao e sua importancia no estabelecimento de diagnostico precoce na diarreia infantil aguda